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Expression of the c-Kit receptor in germ cells of the seminiferous epithelium in rats with hormonal imbalance
reproductive biology 13 (2013) 333–340
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Expression of the c-Kit receptor in germ cells of the seminiferous epithelium in rats with hormonal imbalance Kamila Misiakiewicz *, Agnieszka Kolasa, Anna Kondarewicz, Mariola Marchlewicz, Barbara Wiszniewska Department of Histology and Embryology Pomeranian Medical University in Szczecin, Powstańców Wielkopolskich 72, 70-111 Szczecin, Poland
article info
abstract
Article history:
The aim of the study was to investigate the effects of pharmacologically induced hormonal
Received 20 February 2013
imbalance in adult male rats treated with letrozole and rats exposed to soya isoflavones on
Accepted 11 October 2013
the testicular morphology and c-Kit receptor (c-Kit-R) expression in germ cells. The study
Keywords:
sexual maturity. Morphological and morphometrical analyses were performed on testicular
Seminiferous epithelium
section, and c-Kit-R was identified using immunohistochemistry. In addition, concentration
c-Kit receptor
of circulating steroids was measured in mature rats exposed to soya isoflavones. A signifi-
was conducted during all developmental periods: prenatal period, lactation, youth, and
Hormonal imbalance
cant reduction in testosterone level in rats exposed to soya isoflavones, and the sloughing of
Letrozole
the premature germ cells into the lumen of the seminiferous tubules in the testes of both
Soya isoflavones
groups of rats were observed. Immunohistochemistry showed a decrease in c-Kit-R expression in germ cells of both experimental groups. Morphometric analysis indicated a decreased thickness of the layers occupied by c-Kit-R-positive spermatogonia, and a decreased diameter of the seminiferous tubules in the testes of both experimental groups of animals. In conclusion, the pharmacologically induced reduction of the estradiol level in adult rats and the diminished level of testosterone in rats exposed to soya isoflavones during the prenatal period, lactation and up to maturity caused similar morphological and functional changes associated with the decreased c-Kit-R expression in germ cells in the seminiferous epithelium. These findings demonstrate the importance of the estrogen/androgen balance for normal testicular morphology and spermatogenesis. # 2013 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
1.
Introduction
The seminiferous epithelium is composed of germ cells and postmitotic Sertoli cells. Spermatogenesis takes place in the
epithelium and includes four distinct events: (i) stem cell selfrenewal, which maintains a continuous production of germ cells; (ii) proliferation and differentiation of spermatogonia to increase the number of premeiotic cells; (iii) meiosis of spermatocytes; and (iv) spermiogenesis – the differentiation
* Corresponding author. Tel.: +48 91 4661677; fax: +48 91 4661678. E-mail address: [email protected] (K. Misiakiewicz). 1642-431X/$ – see front matter # 2013 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved. http://dx.doi.org/10.1016/j.repbio.2013.10.004
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of the newly formed oval-shaped spermatids into mature spermatozoa [1]. Spermatogenesis in adult testes is controlled by hormones and other regulatory factors. The interaction of the c-Kit receptor (c-Kit-R) with its ligand SCF (stem cell factor) produced by Sertoli cells plays an essential role in this regulation [1–3]. The expression of c-Kit-R in differentiating spermatogonia and the c-Kit-R/SCF interaction are required for proliferation, differentiation and/or survival of the cells [4]. Because mitosis of differentiating type A spermatogonia (A1– A4) is absolutely dependent on the c-Kit-R/SCF interaction [5], the c-Kit-R/SCF system is essential for male fertility [6]. Despite the fact that c-Kit-R is expressed in the progenitor germ cells (PGCs) during their migration into the genital ridges and in differentiating spermatogonia that arise from the gonocytes of premature testis [1,7], c-Kit-R is also detected in type A differentiating spermatogonia, type B spermatogonia, and premeiotic and meiotic spermatocytes in the adult testes. Moreover, it is specifically expressed in postmeiotic germ cells i.e. elongated spermatids (cytoplasmic droplets, residual bodies, the midpiece of the flagellum) and, at low levels, in round spermatids [1–4,8,9]. It was also demonstrated that cKit-R is expressed in human and animal Leydig cells [9,10]. The proliferation of spermatogonia is androgen- and estrogen-dependent [11]. The blockade of estrogen synthesis by specific aromatase inhibitors is one of the best ways to study estrogen action in vivo [12]. Letrozole is a potent specific non-steroidal third-generation aromatase inhibitor used in clinical therapy and animal experiments [13,14]. Data on males with aromatase deficiency have shown that an undetectable estrogen level is associated with Sertoli cell-only syndrome, hypospermatogenesis, oligoazoospermia, and reduced sperm motility [15–17]. Phytoestrogens, which are categorized as isoflavones, lignans, coumestans, and resorcyclic acid lactones, are nonsteroidal compounds that bind to estrogen receptors (ERs). Lignans and isoflavones are converted in the human digestive tract into heterocyclic phenols, which have a chemical structure similar to those of endogenous mammalian estrogens [18]. Genistein and daidzein are two major soya isoflavone glucosides that are present in high concentrations in soybean and soybean-derived products [19]. Certainly, the influence of isoflavones on the male reproductive system is controversial. Nevertheless, literature data indicate many disturbances in the morphology and function of the testes under the influence of phytoestrogens [20–24]. It has been reported that exposure to exogenous estrogens during fetal and neonatal development can be associated with a series of reproductive abnormalities, such as cryptorchidism, impairment of sperm production, problems with fertility and erectile function, and even the increased probability of testicular cancer [25,26]. Current research focusing on the impact of estrogens and phytoestrogens on male fertility has been motivated by epidemiological data pointing to possible time-related and geographical trends in male reproductive function [27]. Thus, the main aim of this study was to examine the effects of pharmacologically induced hormonal imbalance in adult male rats treated with letrozole and rats exposed to low doses of soya isoflavones on testicular morphology and the c-Kit-R expression in germ cells. The study was conducted during all
developmental periods: prenatal period, lactation, youth and sexual maturity.
2.
Materials and methods
2.1.
Animals
The study was performed on the testes of sexually mature male Wistar rats. The animals were used to generate two models of hormonal imbalance. To create estrogen deficiency (Let), the 3-month-old rats (n = 6 animals) received letrozole (Femara®; Novartis Pharma, Germany), a non-steroidal inhibitor of cytochrome P450 aromatase (P450arom). The inhibitor was administered per os [1 mg/kg body weight (bw)/day] for six months i.e., a total duration of three spermatogeneses in rats). The letrozole was given once per day (in the morning) in the form of small pellet formed from letrozole powder and pressed into a piece of bread. The pellet was served to each experimental rat. The animals willingly ate the pellets from the hand of the person performing the experiment. The rats in the control group (C1; n = 6 animals) received the pellet without letrozole. At the end of experiment, the letrozoletreated rats were nine months old [28]. To create the second model of hormonal imbalance, 3month-old female Wistar rats (n = 4) were kept for one week with sexually mature males (2:1). After one week, they were separated from males, and each female was placed in a separate cage. The pregnant females were divided into control and an experimental groups. The animals had free access to food and water. The experimental females (n = 2) received soya isoflavones individually per os (Meno Stop – HASCO Lek, Poland or SoyaMeno-Terpol, Poland; 20 mg/kg bw/day) from the first day of the experiment until weaning. Soya isoflavones were given once per day (in the morning) in the form of a powder supplement in the feed. Control females (n = 2) received their feed without isoflavones. After separation of the pups from their mothers (day 21), young males of the experimental group (SI; n = 6 animals) were treated with the same dosage of soya isoflavones (20 mg/kg bw/day) for 3 months, until reaching the age of sexual maturity. Young males from the control group (C2; n = 6) were treated similarly, but without soya isoflavones. The animals were terminated under Thiopental anesthesia (120 mg/kg1 bw, i.p., Biochemie GmbH, Austria). The dosages of letrozole and soya isoflavones used in the experiment were based on previously published reports [20,27,29,30]. The experiment was conducted in accordance with Polish Law and with the approval of the ethics committee of the Pomeranian Medical University.
2.2.
Determination of hormone level
Testosterone (T) and E2 levels were measured in heart blood of control and isoflavone exposed rats. The clotted samples of blood were centrifuged, and the sera were collected, frozen, and stored at 20 8C until the analysis. The concentration of T and E2 was measured by the ECLIA (Electrochemiluminescent Immunoassay, Cobas 6000 analyzer, Roche, Switzerland). The measurement range and precision of the T assay were 0.025– 15 ng/mL and 0.1 ng/mL, respectively. The measurement
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range and precision of the E2 assay were 5–4300 pg/mL and 12 pg/mL, respectively. The level of 17b-estradiol was significantly lower in the rats treated with letrozole which was performed previously by us [28].
2.3.
Morphology and immunohistochemistry (IHC)
The testes of control and experimental animals were fixed in Bouin's fluid and embedded in paraffin. For morphological analysis, a series of testis slides (3–5 mm) was stained with the periodic acid-Schiff (PAS) method. To visualize c-Kit-R in germ cells via IHC, rabbit polyclonal anti-c-kit-R antobodies (sc-5535, Santa Cruz Biotechnology, CA, USA) were used. The deparaffinized sections of testes were microwaved in citrate buffer (pH 6.0) for heat-induced epitope retrieval. After slow cooling to room temperature, the slides were washed in PBS twice for 5 min, and then incubated for 60 min with primary anti-c-Kit-R antibody (dilution 1:100). The sections were then stained with an avidin–biotin-peroxidase system, and diaminobenzidine (Dako LSAB + System – HRP: Code K0679 Dako Cytomation, Glostrup, Denmark). The sections were washed in distilled H2O and counterstained with hematoxylin. For a negative control, the specimens were processed in the absence of a primary antibody. A positive staining was defined microscopically by visual identification of the brown pigmentation.
2.4.
Morphometry
For each animal (6/group), two slides with two sections were chosen at random cross sections to obtain the morphometric measurements of the diameter of the seminiferous tubules and the thickness of the layers occupied by c-Kit-R-positive spermatogonia. A total of 720 measurements (30 for each animal) were performed for each parameter. The seminiferous tubules in stages I–V of the cycle of the seminiferous epithelium were chosen and pooled as a single group in morphometric analysis. The stages of the cycle of the seminiferous epithelium were identified according to Leblond and Clermont [31]. The diameters of 720 seminiferous tubules were measured at the shortest distance between two parallel tangent lines of the outer edge of the tubule [32], and the thickness of the layers of c-Kit-R-positive spermatogonia population was measured from the outer edge of the tubule to the nucleus of the primary spermatocyte. All the morphometric measurements were obtained with Axio Vision Rel. 4.6 software (Zeiss, Axioscop, Germany).
2.5.
Statistical analysis
The results were analyzed using Statistica 6.1 software (StatSoft Inc., Tulsa, OK, USA). The arithmetical mean and standard deviation (SD) were calculated for each of the studied parameters. The distribution of the results for the individual variables was obtained by the Shapiro–Wilk test. As most of the distributions deviated from a normal Gaussian distribution, to assess the differences between the studied groups, a non-parametric Mann–Whitney U-test was used. The level of significance was p 0.05.
3.
Results
3.1.
Serum 17b-estradiol and testosterone levels
The serum T level was significantly lower (0.029 0.01 ng/mL) in rats receiving phytoestrogens (SI group) than in control rats (C2 group; 2.1 0.89 ng/mL). There was no difference in serum E2 level between experimental and control rats (Table 1).
3.2.
Morphology of testes
The testes of control rats (C1 and C2 groups) presented a typical morphology. The tubules were lined with seminiferous epithelium containing all generations of germ cells corresponding to the stages of the seminiferous epithelium cycle. The tubular lumen was usually empty (Fig. 1A and B). The letrozole treatment resulted in morphological changes in the seminiferous epithelium. Some tubules showed disorganization of the seminiferous epithelium. In the lumen of the tubules in the experimental rats, clusters of ed germ cells were observed, including late pachytene spermatocytes and spermatids (Fig. 1C). Discontinuous germ cell layers forming irregular intercellular spaces from the effect of the premature germ cell sloughing (Fig. 1C) were also noted. A similar morphology was observed in the testes of the rats exposed to soya isoflavones (Fig. 1D). Sloughed immature germ cells in the lumen of the tubules and empty spaces in the seminiferous epithelium were visible. Additionally, small invaginations of the lamina propria of the tubules were formed (Fig. 1D).
3.3.
Immunoexpression of c-Kit receptor
Immunoexpression of the c-Kit receptor was observed in the cytoplasm of spermatogonia, spermatocytes, and round spermatids (Fig. 2A), as well as in the residual cytoplasm of the elongated spermatids (Fig. 2B) in the seminiferous epithelium of the control rats (C1 and C2 groups). Moreover, a positive reaction was visible in the cytoplasm of Leydig cells in the interstitial tissue (Fig. 2B). The hormonal imbalance caused a decrease or loss of c-Kit-R immunoexpression in the testes cells. A weak expression of c-Kit-R was observed in the
Table 1 – Serum concentrations of testosterone (T) and 17b-estradiol (E2) in control rats (C2) and rats exposed to soya isoflavones (SI). Steroid
C2 group
SI group
T (ng/mL)
M Q1–Q3 Mean SD
1.89 1.45–3.1 2.1 0.98
0.025 0.025–0.025 0.029 0.01*
E2 (pg/mL)
M Q1–Q3 Mean SD
38.37 34.38–49.41 49.3 32.97
31.39 27.33–32.49 30.88 3.52
n = 6 animals/per group; M: median; Q1–Q3: upper quartile and lower quartile; SD: standard deviation. * p < 0.01 (Mann–Whitney U-test).
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Fig. 1 – Cross-section of seminiferous tubules in control rats (A and B), rat receiving letrozole (C), and rat exposed to soya isoflavones (D). All generations of germ cells are present in the seminiferous epithelium. In the lumen of the seminiferous tubules of the letrozole-treated rat (C) and rat exposed to soya isoflavones (D), sloughed immature germ cells (red asterisk) and empty areas in the seminiferous epithelium (red arrows) are visible (C and D). Small invagination of the lamina propria in the seminiferous tubule of a rat exposed to soya isoflavones (black arrow) (D). Periodic acid-Schiff method staining; magnification: 670T.
cytoplasm of spermatogonia, spermatocytes, and spermatids in rats treated with letrozole (Fig. 3A) and those exposed to soya isoflavones (Fig. 3B). A relatively high level of expression of c-Kit-R was visible in the cytoplasmic conglomerations of
residual bodies of spermatozoa in the lumen of seminiferous tubules of the Let group (Fig. 3A). There was very low immunoexpression of c-Kit-R in the interstitial Leydig cells (Fig. 3A and B) in both experimental groups.
Fig. 2 – Immunoexpression of c-Kit-receptor in testes of control rats (A and B). Positive reaction is visible in the cytoplasm of spermatogonia, spermatocytes, and round spermatids (red arrows) (A, and insets A and B) as well as in the residual cytoplasm of the elongated spermatids (green arrows) and in the cytoplasm of the Leydig cells of the interstitial tissue (black arrowheads) (B), (C): negative control. Immunohistochemical staining; magnification: 670T (insets – 670T).
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Fig. 3 – Immunoexpression of c-Kit-receptor in the testes of rats receiving letrozole (A) and rats exposed to soya isoflavones (B). Weak reaction in the cytoplasm of a few spermatogonia, spermatocytes and round spermatids (red arrows) (A and inset, B and inset), as well as in the residual cytoplasm of the elongated spermatids (green arrows) (A and inset, B), in the residual bodies of the spermatozoa in the tubule lumen (green arrows) (A). Low immunopositive reaction in the Leydig cells (black arrowheads) (A and B and inset). Immunohistochemical staining; magnification: 670T (insets – 670T).
3.4.
Seminiferous tubule morphometry
The diameter of the seminiferous tubules in the testes of the Let group was significantly (p 0.05) lower than in respective control rats. Similarly, the thickness of the layers occupied by c-Kit-R-positive spermatogonia in the testes of rats treated with letrozole was lower (p 0.05) than in the testes of rats in the control group (Table 2). A significant decrease (p 0.05) in the diameter of the seminiferous tubules and the thickness of layers with c-Kit-Rpositive spermatogonia was also observed in the testes of rats exposed to soya isoflavones compared to the control rats (Table 3).
4.
Discussion
In the present study, we investigated c-Kit-R expression in germ cells of the seminiferous epithelium of testes in rats with a hormonal imbalance. In the first group of adult rats, the imbalance was pharmacologically induced by letrozole, a nonsteroidal cytochrome P450 aromatase inhibitor. The letrozole
has been proved previously to cause a significant reduction in serum E2 level [28]. In the second group, the rats were prenatally and postnatally exposed to soya isoflavones, which caused a significant decrease in the serum T level. Similar decrease in T level was observed in rats fed with an isoflavone rich diet for 35 days [21] as well as in rats treated with diethylstilbestrol [22]. However, no differences were observed in E2 level in adult rats and in rats treated with phytoestrogens during the perinatal period [21,23]. In the current study, we found that morphology of seminiferous tubules and expression of c-Kit-R in the germ cells were affected by the treatment with letrozole and isoflavones, and were associated with decreased level of serum E2 and T, respectively. Estrogens are involved in the regulation of spermatogenesis and they exert their effects via ERs which are present in the germ cells [33]. The continuous presence of ERs in the seminiferous epithelium indicates that estrogens may directly influence germ cells. It should be emphasized that germ cells are able to synthesize estrogens [34]. In our study, the presence of immature germ cells in the lumen of many seminiferous tubules was a common feature in the rat gonads of both experimental groups. The phenomenon
Table 2 – The diameter of seminiferous tubules and thickness of the c-Kit-R-positive spermatogonia layers of testes in control rats (C1) and letrozole-treated rats (Let). Diameter of seminiferous tubules (mm)
Group
Thickness of the spermatogonia layer (mm)
C1
M Q1–Q3 Mean SD
373.7 324.57–405.61 367.49 59.34
10.75 8.83–12.92 11.09 3.17
Let
M Q1–Q3 Mean SD
312.14 280.55–351.63 312.02 49.21*
7.82 6.51–9.8 8.07 2.45*
n = 6 animals/per group; n = 180/group: number of sections of seminiferous tubules in each group; M: median; Q1–Q3: upper quartile and lower quartile; SD: standard deviation. * p < 0.001 (Mann–Whitney U-test).
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Table 3 – The diameter of seminiferous tubules and thickness of the c-Kit-R-positive spermatogonia of testes in control rats (C2) and rats exposed to soya isoflavones (SI). Group
Diameter of seminiferous tubules (mm)
Thickness of layers of spermatogonia (mm)
C2
M Q1–Q3 Mean SD
307.72 280.29–333.93 310.36 43.50
9.49 8.29–11.08 9.76 2.01
SI
M Q1–Q3 Mean SD
264.44 241.94–279.87 260.68 29.67*
7.18 6.1–8.42 7.25 1.73*
n = 6 animals/per group; n = 180/group: number of sections of seminiferous tubules in each group; M: median; Q1-Q3: upper quartile and lower quartile, SD: standard deviation. * p < 0.001 (Mann–Whitney U-test).
was also observed in our earlier studies on rats with estrogen imbalance [24,28]. It was demonstrated previously that remodeling of intercellular junctions in the seminiferous epithelium during spermatogenesis is regulated by E2, T and cytokines [35]. Estrogens affect the distribution of integral membrane proteins such as occludin, N-cadherin, and connexin 43 via an increase in protein endocytosis, which destabilizes the integrity and function of the seminiferous epithelium [35]. A sloughing of germ cells was previously demonstrated in dihydrotestosterone (DHT) deficiency [36], T level reduction [37] or antiandrogen treatment [38]. The invaginations of basement membrane of seminiferous tubules, another morphological abnormality described both in the current paper and in previous reports [24,28], could result from modifications of anchoring junctions between Sertoli cells and the basement membrane [39]. Therefore, both the invaginations and sloughing of premature germ cells into the lumen of seminiferous tubules observed in the testes of the SI and Let rats may be caused by a hormonal imbalance between androgens and estrogens. The expression of c-Kit-R in the germ cells and Leydig cells of control rats are consistent with earlier reported results. The expression of c-Kit-R was demonstrated in the same cell types in humans [9] and some animal species [3,5,9]. In the current study, we found that the hormonal imbalance was associated with the expression of c-Kit-R in both the SI and Let rats. Immunoexpression of c-Kit-R was lower in germ cells and Leydig cells of experimental than control rats. It was shown that both the estrogen deficiency produced by anti-estrogens and the exposure to estrogens affected testicular tissue, especially during development of the male reproductive system [24,28]. It was demonstrated that in utero exposure of male rats to phthalate esters altered the expression of genes necessary for steroidogenesis and interactions between Sertoli cells and gonocytes [40]. It appears that individual cell types within the testes require a specific hormonal milieu to induce genes necessary for normal functioning of germ cells and other cells implicated in the regulation of spermatogenesis. Cyclins are the principal regulatory proteins involved in the proliferation of germ cells [41]. It is hypothesized that c-Kit-R/ SCF interaction also plays a role in the proliferation and survival of mitotic germ cells in the adult testes [42]. The appropriate ratio between proliferating cells and apoptotic cells is needed for proper spermatogenesis. Apoptosis of germ
cells increased during the first cycle of spermatogenesis and during adult spermatogenesis in mice with KIT (a gene encoding a transmembrane tyrosine-kinase receptor; c-KitR) haplodeficiency or with inactivated allele of the c-Kit-R gene [43]. In addition, the inhibition of the binding of SCF to c-Kit-R promoted mouse spermatogonia apoptosis [44]. Moreover, a decrease in the c-Kit-R expression in the testes of subfertile men was found to be associated with an increased apoptosis in the germ cells [45]. An in vitro study showed that the SCF/c-KitR system up-regulated cyclin D3 and promoted the cell cycle in mouse spermatogonia [46]. The highest level of c-Kit-R was observed in a variety of human testicular pathologies [10], and the overexpression of cyclin E in mouse spermatogonia diminished spermatogonial proliferation and, consequently, reduced fertility [47]. It can be concluded that c-Kit-R acts to prevent apoptosis and promote proliferation of germ cells in the testis. In the current study, we observed a reduction of both the diameter of the seminiferous tubules and thickness of c-Kit-Rpositive spermatogonia layer in both experimental groups when compared to the controls. Similarly, knockout mice with a lack of functional aromatase enzyme (ArKO), although initially fertile, developed progressively disrupted spermatogenesis, and in one-year-old animals, the volume of seminiferous epithelium decreased by 34% [48]. In these mice, disruption of spermatogenesis was characterized by an increased number of apoptotic germ cells, a significant reduction in round and elongated spermatids, and a marked decrease in the volume of seminiferous epithelium [48]. Wistar rats perinatally treated with letrozole displayed a decreased number of testicular spermatozoa and reduced daily sperm production in adult rats [30]. Moreover, in adult male primates, a blockade of estrogen synthesis (by letrozole, fadrozole hydrochloride or CGP 47645) led to the disruption of testicular germ cell transformation, a marked reduction in a number of elongating/elongated spermatids, and consequently, to a significant reduction in sperm cell production [12]. One-year treatment of adult Wistar rats with anastrozole (a nonsteroidal aromatase inhibitor) produced Sertoli cell-only testes in 10% of the animals [14], and it had no effect on the diameter of the seminiferous tubules [49]. Similar effects were observed in adult mice treated with anti-estrogen ICI 182,780 [13] and in animals exposed to soya isoflavones [50,51]. A significant decrease in the adult testicular germ cell volume per testis was observed in rats treated neonatally with diethylstilbestrol
reproductive biology 13 (2013) 333–340
(DES) or ethinyl estradiol [50]. Similarly, the treatment of prepubertal male rats (at postnatal days 5–15) with xenoestrogens (DES, zearalenone) caused detrimental effects on the number of different germ cell types, and reduced the diameter of the seminiferous tubules [51]. In male rats exposed to genistein and vinclozolin from conception to adulthood, the most significant alterations were observed in adult animals. The changes included a reduction in sperm production and motility [52]. Therefore, our results are with accordance with other studies [30,48,50,51]. Moreover, the decreased thickness of c-Kit-R positive spermatogonia layers in our both experimental groups is not surprising, because we also observed decreased expression of c-Kit-R, which is involved in the proliferation of spermatogonia, in the same groups of rats. In conclusion, there is a clear evidence that the estrogen/ androgen balance is important for the maintenance of normal testicular morphology and spermatogenesis. The pharmacologically induced reduction of E2 level in adult rats and the diminished level of T in rats exposed to soya isoflavones caused similar morphological and functional changes associated with a decrease in c-Kit-R expression in germ cells in the seminiferous epithelium.
Conflict of interest The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the article reported.
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[24] Piotrowska K, Baranowska-Bosiacka I, Marchlewicz M, Gutowska I, Noceń I, Zawiślak M, et al. Changes in male reproductive system and mineral metabolism induced by soy isoflavones administered to rats from prenatal life until sexual maturity. Nutrition 2011;27(3):372–9. [25] McLachlan JA. Commentary: prenatal exposure to diethylstilbestrol (DES): a continuing story. International Journal of Epidemiology 2006;35(4):868–70. [26] Gill WB, Schumacher GF, Bibbo M, Straus 2nd FH, Schoenberg HW. Association of diethylstilbestrol exposure in utero with cryptorchidism, testicular hypoplasia and semen abnormalities. The Journal of Urology 1979; 122(1):36–9. [27] Giwercman A. Estrogens and phytoestrogens in male infertility. Current Opinion in Urology 2011;21:519–26. [28] Kondarewicz A, Kolasa A, Zawiślak B, Baranowska-Bosiacka I, Marchlewicz M, Wenda-Różewicka L, et al. Testis morphology in rats chronically treated with letrozole, an aromatase inhibitor. Folia Histochemica et Cytobiologica 2011;49(4):677–84. [29] Degen GH, Janning P, Diel P, Michna H, Bolt HM. Transplacental transfer of the phytoestrogen daidzein in DA/Han rats. Archives of Toxicology 2002;76(1):23–9. [30] Gerardin DC, Pereira OC. Reproductive changes in male rats treated perinatally with an aromatase inhibitor. Pharmacology Biochemistry and Behavior 2002;71(1–2): 301–5. [31] Leblond C, Clermont Y. Definition of the stages of the cycle of the seminiferous epithelium in the rat. Annals of the New York Academy of Sciences 1952;55(4):548–73. [32] Liu Z, Chang Q, Xu ZL, Zhang ZG. Stereological measurement of rat's seminiferous tubule. Chinese Medical Journal 2009;122(21):2643–6. [33] Carreau S, Lambard S, Delalande C, Denis-Galeraud I, Bilińska B, Bourguiba S. Aromatase expression and role of estrogens in male gonad: a review. Reproductive Biology and Endocrinology 2003;1:35–45. [34] Shasa Ch. Estrogens and spermatogenesis. Advances in Experimental Medicine and Biology 2008;636:42–64. [35] Lee NP, Mruk D, Lee WM, Cheng CY. Is the cadherin/catenin protein complex a functional unit of cell-cell actin-based adherens junctions in the rat testis? Biology of Reproduction 2003;68(2):489–508. [36] Kolasa A, Marchlewicz M, Wenda-Różewicka L, Wiszniewska B. Morphology of the testis and the epididymis in rats with dihydrotestosterone (DHT) deficiency. Rocznik Akademii Medycznej w Bialymstoku 2004;49(Suppl. (1)):117–9. [37] Martinez M, Macera S, De Assis GF, Pinheiro PF, Almeida CC, Tirapelli LF, et al. Structural evaluation of the effects of chronic ethanol ingestion on the testis of Calomys callosus. Tissue and Cell 2009;41(3):199–205. [38] Sasso-Cerri E, Cerri P. Morphological evidences indicate that the interference of cimitidine on the peritubular components is responsible for detachment and apoptosis of Sertoli cells. Reproductive Biology and Endocrinology 2008;6:18. [39] Siu MK, Cheng CY. Extracellular matrix: recent advances on its role in the junction dynamics in the seminiferous
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Expression of the c-Kit receptor in germ cells of the seminiferous epithelium in rats with hormonal imbalance Kamila Misiakiewicz *, Agnieszka Kolasa, Anna Kondarewicz, Mariola Marchlewicz, Barbara Wiszniewska Department of Histology and Embryology Pomeranian Medical University in Szczecin, Powstańców Wielkopolskich 72, 70-111 Szczecin, Poland
article info
abstract
Article history:
The aim of the study was to investigate the effects of pharmacologically induced hormonal
Received 20 February 2013
imbalance in adult male rats treated with letrozole and rats exposed to soya isoflavones on
Accepted 11 October 2013
the testicular morphology and c-Kit receptor (c-Kit-R) expression in germ cells. The study
Keywords:
sexual maturity. Morphological and morphometrical analyses were performed on testicular
Seminiferous epithelium
section, and c-Kit-R was identified using immunohistochemistry. In addition, concentration
c-Kit receptor
of circulating steroids was measured in mature rats exposed to soya isoflavones. A signifi-
was conducted during all developmental periods: prenatal period, lactation, youth, and
Hormonal imbalance
cant reduction in testosterone level in rats exposed to soya isoflavones, and the sloughing of
Letrozole
the premature germ cells into the lumen of the seminiferous tubules in the testes of both
Soya isoflavones
groups of rats were observed. Immunohistochemistry showed a decrease in c-Kit-R expression in germ cells of both experimental groups. Morphometric analysis indicated a decreased thickness of the layers occupied by c-Kit-R-positive spermatogonia, and a decreased diameter of the seminiferous tubules in the testes of both experimental groups of animals. In conclusion, the pharmacologically induced reduction of the estradiol level in adult rats and the diminished level of testosterone in rats exposed to soya isoflavones during the prenatal period, lactation and up to maturity caused similar morphological and functional changes associated with the decreased c-Kit-R expression in germ cells in the seminiferous epithelium. These findings demonstrate the importance of the estrogen/androgen balance for normal testicular morphology and spermatogenesis. # 2013 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
1.
Introduction
The seminiferous epithelium is composed of germ cells and postmitotic Sertoli cells. Spermatogenesis takes place in the
epithelium and includes four distinct events: (i) stem cell selfrenewal, which maintains a continuous production of germ cells; (ii) proliferation and differentiation of spermatogonia to increase the number of premeiotic cells; (iii) meiosis of spermatocytes; and (iv) spermiogenesis – the differentiation
* Corresponding author. Tel.: +48 91 4661677; fax: +48 91 4661678. E-mail address: [email protected] (K. Misiakiewicz). 1642-431X/$ – see front matter # 2013 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved. http://dx.doi.org/10.1016/j.repbio.2013.10.004
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of the newly formed oval-shaped spermatids into mature spermatozoa [1]. Spermatogenesis in adult testes is controlled by hormones and other regulatory factors. The interaction of the c-Kit receptor (c-Kit-R) with its ligand SCF (stem cell factor) produced by Sertoli cells plays an essential role in this regulation [1–3]. The expression of c-Kit-R in differentiating spermatogonia and the c-Kit-R/SCF interaction are required for proliferation, differentiation and/or survival of the cells [4]. Because mitosis of differentiating type A spermatogonia (A1– A4) is absolutely dependent on the c-Kit-R/SCF interaction [5], the c-Kit-R/SCF system is essential for male fertility [6]. Despite the fact that c-Kit-R is expressed in the progenitor germ cells (PGCs) during their migration into the genital ridges and in differentiating spermatogonia that arise from the gonocytes of premature testis [1,7], c-Kit-R is also detected in type A differentiating spermatogonia, type B spermatogonia, and premeiotic and meiotic spermatocytes in the adult testes. Moreover, it is specifically expressed in postmeiotic germ cells i.e. elongated spermatids (cytoplasmic droplets, residual bodies, the midpiece of the flagellum) and, at low levels, in round spermatids [1–4,8,9]. It was also demonstrated that cKit-R is expressed in human and animal Leydig cells [9,10]. The proliferation of spermatogonia is androgen- and estrogen-dependent [11]. The blockade of estrogen synthesis by specific aromatase inhibitors is one of the best ways to study estrogen action in vivo [12]. Letrozole is a potent specific non-steroidal third-generation aromatase inhibitor used in clinical therapy and animal experiments [13,14]. Data on males with aromatase deficiency have shown that an undetectable estrogen level is associated with Sertoli cell-only syndrome, hypospermatogenesis, oligoazoospermia, and reduced sperm motility [15–17]. Phytoestrogens, which are categorized as isoflavones, lignans, coumestans, and resorcyclic acid lactones, are nonsteroidal compounds that bind to estrogen receptors (ERs). Lignans and isoflavones are converted in the human digestive tract into heterocyclic phenols, which have a chemical structure similar to those of endogenous mammalian estrogens [18]. Genistein and daidzein are two major soya isoflavone glucosides that are present in high concentrations in soybean and soybean-derived products [19]. Certainly, the influence of isoflavones on the male reproductive system is controversial. Nevertheless, literature data indicate many disturbances in the morphology and function of the testes under the influence of phytoestrogens [20–24]. It has been reported that exposure to exogenous estrogens during fetal and neonatal development can be associated with a series of reproductive abnormalities, such as cryptorchidism, impairment of sperm production, problems with fertility and erectile function, and even the increased probability of testicular cancer [25,26]. Current research focusing on the impact of estrogens and phytoestrogens on male fertility has been motivated by epidemiological data pointing to possible time-related and geographical trends in male reproductive function [27]. Thus, the main aim of this study was to examine the effects of pharmacologically induced hormonal imbalance in adult male rats treated with letrozole and rats exposed to low doses of soya isoflavones on testicular morphology and the c-Kit-R expression in germ cells. The study was conducted during all
developmental periods: prenatal period, lactation, youth and sexual maturity.
2.
Materials and methods
2.1.
Animals
The study was performed on the testes of sexually mature male Wistar rats. The animals were used to generate two models of hormonal imbalance. To create estrogen deficiency (Let), the 3-month-old rats (n = 6 animals) received letrozole (Femara®; Novartis Pharma, Germany), a non-steroidal inhibitor of cytochrome P450 aromatase (P450arom). The inhibitor was administered per os [1 mg/kg body weight (bw)/day] for six months i.e., a total duration of three spermatogeneses in rats). The letrozole was given once per day (in the morning) in the form of small pellet formed from letrozole powder and pressed into a piece of bread. The pellet was served to each experimental rat. The animals willingly ate the pellets from the hand of the person performing the experiment. The rats in the control group (C1; n = 6 animals) received the pellet without letrozole. At the end of experiment, the letrozoletreated rats were nine months old [28]. To create the second model of hormonal imbalance, 3month-old female Wistar rats (n = 4) were kept for one week with sexually mature males (2:1). After one week, they were separated from males, and each female was placed in a separate cage. The pregnant females were divided into control and an experimental groups. The animals had free access to food and water. The experimental females (n = 2) received soya isoflavones individually per os (Meno Stop – HASCO Lek, Poland or SoyaMeno-Terpol, Poland; 20 mg/kg bw/day) from the first day of the experiment until weaning. Soya isoflavones were given once per day (in the morning) in the form of a powder supplement in the feed. Control females (n = 2) received their feed without isoflavones. After separation of the pups from their mothers (day 21), young males of the experimental group (SI; n = 6 animals) were treated with the same dosage of soya isoflavones (20 mg/kg bw/day) for 3 months, until reaching the age of sexual maturity. Young males from the control group (C2; n = 6) were treated similarly, but without soya isoflavones. The animals were terminated under Thiopental anesthesia (120 mg/kg1 bw, i.p., Biochemie GmbH, Austria). The dosages of letrozole and soya isoflavones used in the experiment were based on previously published reports [20,27,29,30]. The experiment was conducted in accordance with Polish Law and with the approval of the ethics committee of the Pomeranian Medical University.
2.2.
Determination of hormone level
Testosterone (T) and E2 levels were measured in heart blood of control and isoflavone exposed rats. The clotted samples of blood were centrifuged, and the sera were collected, frozen, and stored at 20 8C until the analysis. The concentration of T and E2 was measured by the ECLIA (Electrochemiluminescent Immunoassay, Cobas 6000 analyzer, Roche, Switzerland). The measurement range and precision of the T assay were 0.025– 15 ng/mL and 0.1 ng/mL, respectively. The measurement
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range and precision of the E2 assay were 5–4300 pg/mL and 12 pg/mL, respectively. The level of 17b-estradiol was significantly lower in the rats treated with letrozole which was performed previously by us [28].
2.3.
Morphology and immunohistochemistry (IHC)
The testes of control and experimental animals were fixed in Bouin's fluid and embedded in paraffin. For morphological analysis, a series of testis slides (3–5 mm) was stained with the periodic acid-Schiff (PAS) method. To visualize c-Kit-R in germ cells via IHC, rabbit polyclonal anti-c-kit-R antobodies (sc-5535, Santa Cruz Biotechnology, CA, USA) were used. The deparaffinized sections of testes were microwaved in citrate buffer (pH 6.0) for heat-induced epitope retrieval. After slow cooling to room temperature, the slides were washed in PBS twice for 5 min, and then incubated for 60 min with primary anti-c-Kit-R antibody (dilution 1:100). The sections were then stained with an avidin–biotin-peroxidase system, and diaminobenzidine (Dako LSAB + System – HRP: Code K0679 Dako Cytomation, Glostrup, Denmark). The sections were washed in distilled H2O and counterstained with hematoxylin. For a negative control, the specimens were processed in the absence of a primary antibody. A positive staining was defined microscopically by visual identification of the brown pigmentation.
2.4.
Morphometry
For each animal (6/group), two slides with two sections were chosen at random cross sections to obtain the morphometric measurements of the diameter of the seminiferous tubules and the thickness of the layers occupied by c-Kit-R-positive spermatogonia. A total of 720 measurements (30 for each animal) were performed for each parameter. The seminiferous tubules in stages I–V of the cycle of the seminiferous epithelium were chosen and pooled as a single group in morphometric analysis. The stages of the cycle of the seminiferous epithelium were identified according to Leblond and Clermont [31]. The diameters of 720 seminiferous tubules were measured at the shortest distance between two parallel tangent lines of the outer edge of the tubule [32], and the thickness of the layers of c-Kit-R-positive spermatogonia population was measured from the outer edge of the tubule to the nucleus of the primary spermatocyte. All the morphometric measurements were obtained with Axio Vision Rel. 4.6 software (Zeiss, Axioscop, Germany).
2.5.
Statistical analysis
The results were analyzed using Statistica 6.1 software (StatSoft Inc., Tulsa, OK, USA). The arithmetical mean and standard deviation (SD) were calculated for each of the studied parameters. The distribution of the results for the individual variables was obtained by the Shapiro–Wilk test. As most of the distributions deviated from a normal Gaussian distribution, to assess the differences between the studied groups, a non-parametric Mann–Whitney U-test was used. The level of significance was p 0.05.
3.
Results
3.1.
Serum 17b-estradiol and testosterone levels
The serum T level was significantly lower (0.029 0.01 ng/mL) in rats receiving phytoestrogens (SI group) than in control rats (C2 group; 2.1 0.89 ng/mL). There was no difference in serum E2 level between experimental and control rats (Table 1).
3.2.
Morphology of testes
The testes of control rats (C1 and C2 groups) presented a typical morphology. The tubules were lined with seminiferous epithelium containing all generations of germ cells corresponding to the stages of the seminiferous epithelium cycle. The tubular lumen was usually empty (Fig. 1A and B). The letrozole treatment resulted in morphological changes in the seminiferous epithelium. Some tubules showed disorganization of the seminiferous epithelium. In the lumen of the tubules in the experimental rats, clusters of ed germ cells were observed, including late pachytene spermatocytes and spermatids (Fig. 1C). Discontinuous germ cell layers forming irregular intercellular spaces from the effect of the premature germ cell sloughing (Fig. 1C) were also noted. A similar morphology was observed in the testes of the rats exposed to soya isoflavones (Fig. 1D). Sloughed immature germ cells in the lumen of the tubules and empty spaces in the seminiferous epithelium were visible. Additionally, small invaginations of the lamina propria of the tubules were formed (Fig. 1D).
3.3.
Immunoexpression of c-Kit receptor
Immunoexpression of the c-Kit receptor was observed in the cytoplasm of spermatogonia, spermatocytes, and round spermatids (Fig. 2A), as well as in the residual cytoplasm of the elongated spermatids (Fig. 2B) in the seminiferous epithelium of the control rats (C1 and C2 groups). Moreover, a positive reaction was visible in the cytoplasm of Leydig cells in the interstitial tissue (Fig. 2B). The hormonal imbalance caused a decrease or loss of c-Kit-R immunoexpression in the testes cells. A weak expression of c-Kit-R was observed in the
Table 1 – Serum concentrations of testosterone (T) and 17b-estradiol (E2) in control rats (C2) and rats exposed to soya isoflavones (SI). Steroid
C2 group
SI group
T (ng/mL)
M Q1–Q3 Mean SD
1.89 1.45–3.1 2.1 0.98
0.025 0.025–0.025 0.029 0.01*
E2 (pg/mL)
M Q1–Q3 Mean SD
38.37 34.38–49.41 49.3 32.97
31.39 27.33–32.49 30.88 3.52
n = 6 animals/per group; M: median; Q1–Q3: upper quartile and lower quartile; SD: standard deviation. * p < 0.01 (Mann–Whitney U-test).
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Fig. 1 – Cross-section of seminiferous tubules in control rats (A and B), rat receiving letrozole (C), and rat exposed to soya isoflavones (D). All generations of germ cells are present in the seminiferous epithelium. In the lumen of the seminiferous tubules of the letrozole-treated rat (C) and rat exposed to soya isoflavones (D), sloughed immature germ cells (red asterisk) and empty areas in the seminiferous epithelium (red arrows) are visible (C and D). Small invagination of the lamina propria in the seminiferous tubule of a rat exposed to soya isoflavones (black arrow) (D). Periodic acid-Schiff method staining; magnification: 670T.
cytoplasm of spermatogonia, spermatocytes, and spermatids in rats treated with letrozole (Fig. 3A) and those exposed to soya isoflavones (Fig. 3B). A relatively high level of expression of c-Kit-R was visible in the cytoplasmic conglomerations of
residual bodies of spermatozoa in the lumen of seminiferous tubules of the Let group (Fig. 3A). There was very low immunoexpression of c-Kit-R in the interstitial Leydig cells (Fig. 3A and B) in both experimental groups.
Fig. 2 – Immunoexpression of c-Kit-receptor in testes of control rats (A and B). Positive reaction is visible in the cytoplasm of spermatogonia, spermatocytes, and round spermatids (red arrows) (A, and insets A and B) as well as in the residual cytoplasm of the elongated spermatids (green arrows) and in the cytoplasm of the Leydig cells of the interstitial tissue (black arrowheads) (B), (C): negative control. Immunohistochemical staining; magnification: 670T (insets – 670T).
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Fig. 3 – Immunoexpression of c-Kit-receptor in the testes of rats receiving letrozole (A) and rats exposed to soya isoflavones (B). Weak reaction in the cytoplasm of a few spermatogonia, spermatocytes and round spermatids (red arrows) (A and inset, B and inset), as well as in the residual cytoplasm of the elongated spermatids (green arrows) (A and inset, B), in the residual bodies of the spermatozoa in the tubule lumen (green arrows) (A). Low immunopositive reaction in the Leydig cells (black arrowheads) (A and B and inset). Immunohistochemical staining; magnification: 670T (insets – 670T).
3.4.
Seminiferous tubule morphometry
The diameter of the seminiferous tubules in the testes of the Let group was significantly (p 0.05) lower than in respective control rats. Similarly, the thickness of the layers occupied by c-Kit-R-positive spermatogonia in the testes of rats treated with letrozole was lower (p 0.05) than in the testes of rats in the control group (Table 2). A significant decrease (p 0.05) in the diameter of the seminiferous tubules and the thickness of layers with c-Kit-Rpositive spermatogonia was also observed in the testes of rats exposed to soya isoflavones compared to the control rats (Table 3).
4.
Discussion
In the present study, we investigated c-Kit-R expression in germ cells of the seminiferous epithelium of testes in rats with a hormonal imbalance. In the first group of adult rats, the imbalance was pharmacologically induced by letrozole, a nonsteroidal cytochrome P450 aromatase inhibitor. The letrozole
has been proved previously to cause a significant reduction in serum E2 level [28]. In the second group, the rats were prenatally and postnatally exposed to soya isoflavones, which caused a significant decrease in the serum T level. Similar decrease in T level was observed in rats fed with an isoflavone rich diet for 35 days [21] as well as in rats treated with diethylstilbestrol [22]. However, no differences were observed in E2 level in adult rats and in rats treated with phytoestrogens during the perinatal period [21,23]. In the current study, we found that morphology of seminiferous tubules and expression of c-Kit-R in the germ cells were affected by the treatment with letrozole and isoflavones, and were associated with decreased level of serum E2 and T, respectively. Estrogens are involved in the regulation of spermatogenesis and they exert their effects via ERs which are present in the germ cells [33]. The continuous presence of ERs in the seminiferous epithelium indicates that estrogens may directly influence germ cells. It should be emphasized that germ cells are able to synthesize estrogens [34]. In our study, the presence of immature germ cells in the lumen of many seminiferous tubules was a common feature in the rat gonads of both experimental groups. The phenomenon
Table 2 – The diameter of seminiferous tubules and thickness of the c-Kit-R-positive spermatogonia layers of testes in control rats (C1) and letrozole-treated rats (Let). Diameter of seminiferous tubules (mm)
Group
Thickness of the spermatogonia layer (mm)
C1
M Q1–Q3 Mean SD
373.7 324.57–405.61 367.49 59.34
10.75 8.83–12.92 11.09 3.17
Let
M Q1–Q3 Mean SD
312.14 280.55–351.63 312.02 49.21*
7.82 6.51–9.8 8.07 2.45*
n = 6 animals/per group; n = 180/group: number of sections of seminiferous tubules in each group; M: median; Q1–Q3: upper quartile and lower quartile; SD: standard deviation. * p < 0.001 (Mann–Whitney U-test).
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Table 3 – The diameter of seminiferous tubules and thickness of the c-Kit-R-positive spermatogonia of testes in control rats (C2) and rats exposed to soya isoflavones (SI). Group
Diameter of seminiferous tubules (mm)
Thickness of layers of spermatogonia (mm)
C2
M Q1–Q3 Mean SD
307.72 280.29–333.93 310.36 43.50
9.49 8.29–11.08 9.76 2.01
SI
M Q1–Q3 Mean SD
264.44 241.94–279.87 260.68 29.67*
7.18 6.1–8.42 7.25 1.73*
n = 6 animals/per group; n = 180/group: number of sections of seminiferous tubules in each group; M: median; Q1-Q3: upper quartile and lower quartile, SD: standard deviation. * p < 0.001 (Mann–Whitney U-test).
was also observed in our earlier studies on rats with estrogen imbalance [24,28]. It was demonstrated previously that remodeling of intercellular junctions in the seminiferous epithelium during spermatogenesis is regulated by E2, T and cytokines [35]. Estrogens affect the distribution of integral membrane proteins such as occludin, N-cadherin, and connexin 43 via an increase in protein endocytosis, which destabilizes the integrity and function of the seminiferous epithelium [35]. A sloughing of germ cells was previously demonstrated in dihydrotestosterone (DHT) deficiency [36], T level reduction [37] or antiandrogen treatment [38]. The invaginations of basement membrane of seminiferous tubules, another morphological abnormality described both in the current paper and in previous reports [24,28], could result from modifications of anchoring junctions between Sertoli cells and the basement membrane [39]. Therefore, both the invaginations and sloughing of premature germ cells into the lumen of seminiferous tubules observed in the testes of the SI and Let rats may be caused by a hormonal imbalance between androgens and estrogens. The expression of c-Kit-R in the germ cells and Leydig cells of control rats are consistent with earlier reported results. The expression of c-Kit-R was demonstrated in the same cell types in humans [9] and some animal species [3,5,9]. In the current study, we found that the hormonal imbalance was associated with the expression of c-Kit-R in both the SI and Let rats. Immunoexpression of c-Kit-R was lower in germ cells and Leydig cells of experimental than control rats. It was shown that both the estrogen deficiency produced by anti-estrogens and the exposure to estrogens affected testicular tissue, especially during development of the male reproductive system [24,28]. It was demonstrated that in utero exposure of male rats to phthalate esters altered the expression of genes necessary for steroidogenesis and interactions between Sertoli cells and gonocytes [40]. It appears that individual cell types within the testes require a specific hormonal milieu to induce genes necessary for normal functioning of germ cells and other cells implicated in the regulation of spermatogenesis. Cyclins are the principal regulatory proteins involved in the proliferation of germ cells [41]. It is hypothesized that c-Kit-R/ SCF interaction also plays a role in the proliferation and survival of mitotic germ cells in the adult testes [42]. The appropriate ratio between proliferating cells and apoptotic cells is needed for proper spermatogenesis. Apoptosis of germ
cells increased during the first cycle of spermatogenesis and during adult spermatogenesis in mice with KIT (a gene encoding a transmembrane tyrosine-kinase receptor; c-KitR) haplodeficiency or with inactivated allele of the c-Kit-R gene [43]. In addition, the inhibition of the binding of SCF to c-Kit-R promoted mouse spermatogonia apoptosis [44]. Moreover, a decrease in the c-Kit-R expression in the testes of subfertile men was found to be associated with an increased apoptosis in the germ cells [45]. An in vitro study showed that the SCF/c-KitR system up-regulated cyclin D3 and promoted the cell cycle in mouse spermatogonia [46]. The highest level of c-Kit-R was observed in a variety of human testicular pathologies [10], and the overexpression of cyclin E in mouse spermatogonia diminished spermatogonial proliferation and, consequently, reduced fertility [47]. It can be concluded that c-Kit-R acts to prevent apoptosis and promote proliferation of germ cells in the testis. In the current study, we observed a reduction of both the diameter of the seminiferous tubules and thickness of c-Kit-Rpositive spermatogonia layer in both experimental groups when compared to the controls. Similarly, knockout mice with a lack of functional aromatase enzyme (ArKO), although initially fertile, developed progressively disrupted spermatogenesis, and in one-year-old animals, the volume of seminiferous epithelium decreased by 34% [48]. In these mice, disruption of spermatogenesis was characterized by an increased number of apoptotic germ cells, a significant reduction in round and elongated spermatids, and a marked decrease in the volume of seminiferous epithelium [48]. Wistar rats perinatally treated with letrozole displayed a decreased number of testicular spermatozoa and reduced daily sperm production in adult rats [30]. Moreover, in adult male primates, a blockade of estrogen synthesis (by letrozole, fadrozole hydrochloride or CGP 47645) led to the disruption of testicular germ cell transformation, a marked reduction in a number of elongating/elongated spermatids, and consequently, to a significant reduction in sperm cell production [12]. One-year treatment of adult Wistar rats with anastrozole (a nonsteroidal aromatase inhibitor) produced Sertoli cell-only testes in 10% of the animals [14], and it had no effect on the diameter of the seminiferous tubules [49]. Similar effects were observed in adult mice treated with anti-estrogen ICI 182,780 [13] and in animals exposed to soya isoflavones [50,51]. A significant decrease in the adult testicular germ cell volume per testis was observed in rats treated neonatally with diethylstilbestrol
reproductive biology 13 (2013) 333–340
(DES) or ethinyl estradiol [50]. Similarly, the treatment of prepubertal male rats (at postnatal days 5–15) with xenoestrogens (DES, zearalenone) caused detrimental effects on the number of different germ cell types, and reduced the diameter of the seminiferous tubules [51]. In male rats exposed to genistein and vinclozolin from conception to adulthood, the most significant alterations were observed in adult animals. The changes included a reduction in sperm production and motility [52]. Therefore, our results are with accordance with other studies [30,48,50,51]. Moreover, the decreased thickness of c-Kit-R positive spermatogonia layers in our both experimental groups is not surprising, because we also observed decreased expression of c-Kit-R, which is involved in the proliferation of spermatogonia, in the same groups of rats. In conclusion, there is a clear evidence that the estrogen/ androgen balance is important for the maintenance of normal testicular morphology and spermatogenesis. The pharmacologically induced reduction of E2 level in adult rats and the diminished level of T in rats exposed to soya isoflavones caused similar morphological and functional changes associated with a decrease in c-Kit-R expression in germ cells in the seminiferous epithelium.
Conflict of interest The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the article reported.
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