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- Original Article -

17beta-estradiol stimulates proliferation of spermatogonia in experimental cryptorchid mice

En-Zhong Li^1,2, De-Xue Li¹, Shi-Qing Zhang^1,3, Chang-Yong Wang¹, Xue-Ming Zhang³, Jing-Yan Lu¹, Cui-Mi Duan¹, Xiang-Zhong Yang⁴, Li-Xin Feng⁴

¹Beijing Institute of Basic Medical Sciences, Beijing 100850, China
²Department of Agriculture and Forestry, Huanghuai University, Zhumadian 463000, China
³College of Animal Sciences and Veterinary Medicine, Jilin University, Changchun 130002, China
⁴Department of Animal Science and Center for Regenerative Biology, University of Connecticut, Storrs, Connecticut 06269-4243, USA

Abstract

Aim: To investigate whether estrogen stimulates the proliferation of spermatogonia or induces spermatogenesis in cryptorchid mice.  Methods: Mice were surgically rendered cryptorchid, then treated with different doses of 17β-estradiol (E2) s.c. once a day. Mice were killed at sexual maturity (45 days of age), and histological analysis and immunofluorescence were performed. Serum follicle stimulating hormone (FSH), estradiol, testosterone and luteinizing hormone (LH) were measured.  Results: Low doses of E2 had no notable effect on spermatogonia, but at higher doses, E2 stimulated the proliferation of spermatogonia.  Conclusion: E2 has a dose-related mitogenic effect on spermatogonia. (Asian J Androl 2007 Sep; 9: 659-667)

Keywords: 17β-estradiol; cryptorchid mice; proliferation; spermatogonia

Correspondence to: Dr De-Xue Li, Office of Beijing Academy of Medical Sciences, 27 Taiping Road, Beijing 100850, China.
Tel: +86-10-6693-1009 Fax: +86-10-6693-0055
E-mail: lidx@nic.bmi. ac.cn
Received 2006-08-15 Accepted 2007-04-05

DOI: 10.1111/j.1745-7262.2007.00288.x

1 Introduction

Millions of spermatozoa are produced daily in the mammalian testis from spermatogonial stem cells (SSC) and the germ line stem cells in the testis. To maintain normal spermatogenesis, the balance of self-renewal and differentiation of SSC must be precisely regulated by intrinsic gene expression in the stem cells and extrinsic signals, including soluble factors or adhesion molecules, from the surrounding microenvironment, the stem cell niche [1, 2]. SSC are a kind of unique cell in postnatal animals that both undergoes self-renewal and contributes genes to subsequent generations. Therefore, SSC are essential for species continuity. SSC are also valuable for genome modification and production of transgenic animals. However, SSC are rare in testis tissue, comprising approximately 1 in 5 000 cells in adult mouse testis [1]. Enrichment and purification of SSC are vital to obtain sufficient numbers of cells for study. Therefore, if we could find a better method of obtaining SSC, we could more readily study the cells' development and spermatogenesis. Cryptorchid mice make a likely model for studying spermatogonia in vivo as spermatogenesis is arrested and the spermatogonia are enriched in the spermtogenic epithelium [3]. Estrogen decreases the rate of apoptosis and stimulates proliferation of human spermatogonia in vitro [4], and that of rat spermatogonia in vivo [5, 6], and induces the renewal of SSC in Japanese eels, Anguilla Japonica, in vitro [7] and in lizards (Podarcis S. Sicula) in vivo [8]. We hypothesize that we can study the effects of 17β-estradiol (E2) stimulation of spermatogonia in cryptorchid mice. If E2 stimulates renewal of spermatogonia in cryptorchid mice, then these mice could serve as a source of enriched and expanded spermatogonia for studies on the mechanisms controlling spermatogonia proliferation and differentiation.

2 Materials and methods

2.1 Animals

For the present study, fourty 10-day-old male and eight 45-day-old male Kunming mice were obtained from the Experimental Animal Center of the Academy of Military Medical Sciences (Beijing, China). They were maintained under standard mouse laboratory conditions in a temperature-controlled and light-controlled room (temperature 23 ± 2ºC; 12 h:8 h Light:Dark cycle [lighted approximately 7:00 to 19:00]). The 10-day-old mice remained with their mothers and were allowed to nurse ad libitum. The 45-day-old mice were also kept in the controlled environment with free access to food and water. The experimental protocol was approved by the Animal Experimental Committee of the Beijing Academy of Medical Sciences (Beijing, China).

2.2 Experimental cryptorchidism

The 10-day-old male KM mice were surgically rendered cryptorchid via a modification of a procedure reported by Nishimune and colleagues [9]. A midline lower abdominal incision was made under ether anesthesia. The gubernacula were bilaterally cut and the adipose tissue of the capita epididymidum on each side was sutured to the inner peritoneal wall, pulling the testes into the abdomen. Eight of the 10-day-old mice and all of the 45-day-old mice remained as intact controls.

2.3 Hormone treatment

Surgical cryptorchid mice were randomly divided into four groups of 8 mice each and were treated with E2 (Sigma-Aldrich, St. Louis, MO, USA) dissolved in olive oil at different dosages s.c. Group I received 0.25 µg/g body weight (bw) of E2 (low-estradiol-treated group) per day; Group II, 0.5 µg/g bw (mid-estradiol-treated group) per day; and Group III, 1 µg/g bw (high-estradiol-treated group) per day. Group IV was the control, and was given 10 µL/mouse of olive oil (vehicle only) per day. Each mouse was injected once a day, between 8:00 and 9:00, commencing just after the operation.

2.4 Hormone measurement

The animals were killed with sodium pentobarbital on post-surgical day 35, and blood was collected by removing an eyeball. The serum was separated and stored at _20ºC until analyses of follicle stimulating hormone (FSH), estradiol, testosterone and luteinizing hormone (LH) level could be done. Levels of serum FSH and LH were measured using an enyzme-linked immunosorbent assay (ELISA) kits (RapidBio, West Hills, CA, USA). Intra-assay and inter-assay coefficients of variation for FSH were not more than 1% and were not more than 2% for LH. Serum estradiol and testosterone were measured using the Automated Chemiluminescence System (Bayer, New York, USA). The assay kits were also purchased from Bayer. The sample number is 6_8 for each examination.

2.5 Inhibin B measurement

At the time of death, the right testes of the mice were removed, weighed and stored in liquid nitrogen. They was then homogenized in 0.5 mL deionized water using a Teflon homogenizer that fits into a microfuge tubule. Each sample was centrifuged at 4ºC (9 000 × g, 10 min). The supernatant was frozen for later assay of inhibin B. Plasma and testis concentrations of inhibin B were determined by a two-site ELISA, as previously described [10], with a sensitivity of 30 ng inhibin B/mL and intra-assay and inter-assay coefficients of variation of 4.2% and 9.8%, respectively (n = 6_8). The kit was purchased from Diagnostic Systems Laboratories (Webster, TX, USA).

2.6 Histological analysis

After the right testes were removed, the experimental cryptorchid mice were perfused with Bouin's fluid, and the left testes samples were fixed for 12 h in Bouin's fluid. After washing with ethanol (50%) for three times (30 min each time), the samples were dehydrated, embedded and cut into 6 µm sections at 60 µm intervals. The 6 µm sections were divided into two parts and every other formed into one part. One part was used for hematoxylin eosin (HE), and the other for immunofluorescence analysis. The slides in part I were deparaffinized and hydrated in a graded ethanol series. Slides were stained with HE. If one of the testes had re-descended into the scrotum, neither testis was analyzed histologically. In addition, the eight intact 10-day-old mice and the eight intact 45-day-old mice were killed, and their testes were subjected to the same analyses as the experimental and control (Group IV) groups' testes.

2.7 Immunofluorescence

Both Thy-1 and integrin-β1 are surface markers of SSC in mouse testes [11, 12, 14]. Therefore, immunofluorescence with the two molecules as antigens was performed to identify spermatogonia. The 6-µm slides in Group II were analyzed via immunofluorescence. Deparaffinized sections were hydrated in a graded ethanol series and 0.3% hydrogen peroxide for 20 min to block endogenous peroxidase activity. Antigen was retrieved by microwave oven in 10 mmol/L citrate buffer (pH 6.0), at 95_98ºC for 15 min, and were then treated with 1% normal goat serum for 30 min at 37ºC. The primary antibody (anti-Thy-1 monoclonal antibody, rat anti-mouse [Lab Vision, Fremont, CA, USA] used at a 1:200 dilution and anti-integrin-β1 polyclonal antibody, rabbit anti-mouse [Boster, Wuhan, Hubei, China] used at a 1:200 dilution) was incubated with the sections at 4ºC overnight (approximately 18 h). After washing in phosphate buffered saline (PBS) (0.01 mol/mL), the sections were incubated with FITC-goat-anti-rat and Cy3-goat-anti-rabbit immunoglobulin (Boster, Wuhan, Hubei, China) for 20 min each at 37ºC. The sections were again washed with PBS (0.01 mol/mL) for three times, and once for 5 min and observed under fluorescence microscopy. The negative control sections were treated using the same procedure as above, except that the primary antibody was replaced by PBS. The numbers of Thy-1⁺ and integrin-β1⁺ cells per 100 µm tubule width and per 100 µm tubule perimeter were counted, respectively. The numbers of positive cells were all expressed as number/100 µm tubule width plus number/100 µm tubule perimeter.

2.8 Statistical analysis

The results of number/tubule of Thy-1⁺ and integrin-β1⁺ cells were both expressed as mean ± SD. Serum FSH, estradiol, testosterone and LH concentration and inhibin B level were also expressed as mean ± SD. Statistical comparison between means was analyzed by one-way analysis of variance.

3 Results

3.1 Histological analysis

On histological analysis, testes from 45-day-old mice contained germ cells at every stage of development, from SSC to sperm. This indicates that the animals were fully sexually mature at that age [11]. The degree of differentiation of germ cells was much lower in 10-day-old mice than that in 45-day-old mice; in 10-day-old mice only spermatogonia and primary spermatocytes were present. Furthermore, the spermatogonia were relatively greater in number. In the experimental cryptorchid mice (including estradiol-treated groups and the control group), the types of germ cells were similar to those found in 10-day-old mice. The proportions of spermatogonia to all germ cells in the seminiferous tubules differed in the animals in the various cryptorchid groups. The proportions found in Group IV and in Group I were similar to those found in 10-day-old mice. Comparison among the various estradiol-treated cryptorchid groups showed that the number of spermatogonia present in the testis increased in the presence of estrogen in a dose-related manner. Mice treated with a high dose of E2 had the most testis cells that appeared morphologically similar to spermatogonia (Figure 1).

3.2 Immunofluorescence

Immunofluorescence examination with the Thy-1 antigen showed that the number of cells that reacted to Thy-1 antibody (Thy-1⁺ cells) increased directly with the dose of E2. Group II and Group III both had more than one layer of Thy-1⁺ cells. In contrast, only the cells along the basal membrane of the seminiferous tubules were Thy-1⁺ in 45-day-old intact mice. In 10-day-old mice, Thy-1⁺ cells were also present only along the seminiferous tubules' basal membrane; however, there were more of those cells in 45-day-old mice (Figure 2). Mice in groups I and IV were similar to 10-day-old mice. In addition, immunofluorescence examination with the integrin-β1 marker yielded similar results (Figure 2). In the negative control, peritubular cells were also immunostained, both for Thy-1 and for integrin-β1. These might result from unspecific staining.

Statistical analysis showed significant increases in the number of Thy-1⁺ and integrin-β1⁺ cells as the dose of exogenous estradiol increased (Table 1). Table 1 illustrates the relationship between the amount of estradiol given and the numbers of Thy-1⁺ and integrin-β1⁺ cells present.

3.3 Hormone level

To further understand the endocrine basis of these changes, serum FSH, estradiol, testosterone and LH were measured. As shown in Figure 3, the mature mice (45-day-old mice) had the lowest FSH levels (3.95 ± 1.65 ng/mL); in comparison, serum FSH of mice in Group IV (control group), which was up to 19.1 ± 8.8 ng/mL, was significantly higher (P < 0.05). There were no significant difference in FSH level between any two of the 10-day mice, Group II (38.6 ± 9.2 ng/mL, 45.7 ± 8.1 ng/mL) and Group III (41.5 ± 10.3 ng/mL). However, these levels were all significantly higher than those for Group IV (P < 0.05). Animals treated with a low dose of E2 showed FSH concentrations (25.3 ± 7.3 ng/mL) approximately the same as those of the control group (P = 0.11). LH was not significantly different between any two of the different treated groups (results not shown). Estradiol was significantly different between any two of the E2-treated groups (P < 0.05). Testosterone levels descended with increasing doses of estradiol (Table 2). Serum testosterone levels of mice in Groups II and III were not different from each other, but both higher than those of Group I (P < 0.05), and lower than the control (Group IV) (P < 0.05).

3.4 Inhibin B measurement

The results of the ELISA for testis inhibin B are presented in Figure 4. The plasma inhibin B of normal mature mice (45-day-old) was the highest (409 ± 36 pg/mL), whereas the levels in 10-day-old mice and mid-dose and high-dose E2-treated mice were the lowest (176 ± 30 pg/mL, 119 ± 16 pg/mL and 134 ± 17 pg/mL, respectively) and did not significantly different from each other. Group I (199 ± 21 pg/mL) had higher levels of plasma inhibin B than Group II and Group III (P < 0.05), but no higher level than that the 10-day-old mice had. Inhibin B was higher than any estradiol-treated cryptorchid group (P  < 0.05) in olive oil-treated cryptorchid mice (226 ± 28 pg/mL). On the other hand, serum inhibin B levels were not different between any two of the surgical cryptorchid groups (E2- treated groups and the control group), but were lower than that in 45-day-old mice (results not shown).

4 Discussion

A scrotal temperature approximately 5_7ºC lower than abdominal temperature is required for normal spermatogenesis [12]. Cryptorchidism has detrimental effects on spermatogenesis [12]. In the present study, cryptorchidism showed a striking influence on testis development in KM mice. In these mice, cryptorchidism led to spermatogenesis being arrested at the primary spermatocyte stage (Figure 1D).

In men, estrogen is secreted by Sertoli and Leydig cells, as well as by the adrenal gland. Estrogen receptors are expressed in the male reproductive organ. Estrogen is important for male reproduction [13]. In mammals, it is generally thought that estrogen appears to regulate the synthesis of testosterone and sexual behavior, in addition to spermatogenesis [14]. In recent years, there have been increasingly frequent reports investigating the functions of estrogen in the testis, but the actual effects of estrogen in male reproduction still remain unknown. One of interesting aspects of estrogen function is its role in the regulation of spermatogonia proliferation. Therefore, it struck us as logical to study the role of E2 on SSC development. The numbers and types of germ cells found were similar in Groups I and IV (control). This indicates that low doses of E2 have no remarkable effect on proliferation and differentiation of germ cells in cryptorchid mice. However, the number of germ cells in Groups II and III were both much greater than in Group IV. The increase in cell number was in direct proportion to the dosage of exogenous E2. This suggests that E2 promotes germ cell division in a dose-related fashion.

We used Thy-1 antibody for immunofluorescence examination of the testis to determine if E2 can stimulate spermatogonia proliferation. Thy-1 (CD90) was first identified on the T-cell surface in the thymus. Recent studies have found that Thy-1 is also a unique surface marker of SSC in mouse testes [15_17]. In the present study, more cells in the seminiferous tubules of surgical cryptorchid mice treated with estradiol were Thy-1⁺ cells, especially in the mid-dose and high-dose-estradiol-treated groups. Furthermore, comparison of HE staining (Figure 1F) and immunofluorescence (Figure 2C) of the same field showed that in the 45-day-old mice, only the cells along the basal membrane of the seminiferous tubules were Thy-1⁺. Therefore, it appears that high doses of E2 can promote spermatogonia proliferation.

Reports have established that integrin-β1 is the surface marker on mouse SSC [16, 18]. Therefore, in testis seminiferous tubules, the integrin-β1⁺ cells are SSC. To further confirm that E2 stimulates SSC division toward mitosis, but not meiosis, immunofluorescence was conducted using integrin-β1 as a marker. The results showed that the Thy-1⁺ cells were also integrin-β1⁺. In the present study, the Thy-1⁺ and integrin-β1⁺ cells were regard as spermatogonia.

According to this study, the most effective doses of E2 for producing spermatogonia are 0.5_1.0 µg/g bw. Our data are consistent with those previously reported by Pentikainen et al. [4] and Li et al. [19], in which E2 was observed to stimulate proliferation of spermatogonia and gonocytes in vitro, and a report by Miura et al. [7], in which they found that 10 pg/mL of E2 was sufficient to induce SSC division in cultured testicular tissue.

Proliferation and differentiation of SSC is a complex process that is tightly controlled by both endocrine and paracrine factors. The mechanisms by which estrogen induces the renewal of SSC are not well understood. Estrogen regulation may occur indirectly at the pituitary level by regulating gonadotropin (FSH and LH) secretion. FSH and LH are the primary tropic hormones that regulate testicular function. FSH is well known to regulate spermatogonia; for instance, FSH has been shown to increase the number of spermatogonia in hypogonadal (hpg) mice [20]. Stimulation of FSH by estradiol treatment has been noted in normal and transgenic rodents, notably hpg mice [14].

Inhibins are a group of glycoprotein hormones secreted by the gonads in men and women. The inhibin α subunit heterodimerizes with inhibin b subunits to form inhibin A (α and β_A) and inhibin B (α and β_B), which are the bioactive forms of inhibin in the general circulation. These bioactive forms have been well-characterized as negatively regulating pituitary FSH secretion through direct action on the pituitary gonadotrophs in mature female and male rats [21].

To elucidate the mechanism by which E2 stimulates SSC in surgical cryptorchid mice, we measured serum FSH and plasma and testis inhibin B. Results showed that FSH in surgical cryptorchid mice (including E2-treated groups and the control group) were higher than in normal, 45-day-old mice. This suggests that cryptorchidism causes an increase in serum FSH. There were more spermatogonia (Thy-1⁺ and integrin-β1⁺) in all of these cryptorchid groups than in normal mature mice. This indicates that FSH induces spermatogonial proliferation in cryptorchid mice, consistent with the observation in hypogonadal mice [18]. However, a statistical analysis of FSH level and spermatogonia in the estradiol-treated groups showed that spermatogonia present in the testis increased in a dose-related manner, but FSH level did not. When compared, Group II and III FSH levels (45.7 ± 8.1 ng/mL vs. 41.5 ± 10.3 ng/mL) were not significantly different; but their number of Thy-1⁺ cells (11.2 ± 0.5 and 14.3 ± 0.3, respectively) and integrin-β1⁺ cells (12.0 ± 0.3 and 14.6 ± 0.2, respectively) differed significantly (P < 0.05). Plasma inhibin B levels were not different among the cryptorchid groups, although testis inhibin B levels were different. In addition, we found that estradiol has no significant effect on serum LH (result not shown). These findings suggest that one, but not all, of the pathways by which E2 regulates SSC is via FSH.

In the present study, we found that estrogen inhibits testosterone secretion. This finding suggests that another pathway through which E2 promotes spermatogonia proliferation is by estrogen inhibiting the effect of testosterone on SSC differentiation [22].

There might be two other pathways through which estrogen stimulates SSC proliferation. The first possible pathway concerns the complex of estrogen and its receptors. The estrogen receptor family is composed of ERα and ERβ. The bulk of reports point out that ERα is expressed in Leydig cells and peritubular myoid cells, whereas ERβ is present in both Sertoli cells and germ cells [23, 24]. Subsequently, experimental evidence shows that ERβ regulates germ cell development [25]. ERβ has been shown to mediate estrogen action in much the same way as ERα [26]. Therefore, estrogen might bind to ERβ to activate the genes that promote SSC self-renewal.

The second pathway relates to the ability of estrogen to induce expression of growth factors that stimulate SSC self-renewal. Because estrogen has the ability to bind and activate ERs, it affects testicular gene expression [27]. Possibly, the complex of estrogen and its receptor (ERα) induces Sertoli or Leydig cells to secrete growth factors, such as glial cell derived neurotrophic factor (GDNF), the same way that stem cell factor is produced in Sertoli cells when induced by FSH [28]. Perhaps this is just like the way that E2 stimulates GDNF expression in hypothalamic neurons [29]. In cryptorchid mice, this possible pathway has been implicated in the restoration of spermatogenesis [30]. Furthermore, bovine serum albumin-conjugated E2 decreases testicular androgen production in vitro [31].

All results above indicate that E2 can stimulate spermatogonia proliferation in cryptorchid mice. Treatment of cryptorchid mice with 0.5 µg/g bw and 1.0 µg/g bw of E2 can be used to enrich spermatogonia and produce mice that have more spermatogonia. The mechanism by which E2 stimulate spermatogonia are complex, in which many factors are involved.

Acknowledgment

This work was supported by the National Natural Science Foundation of China (No. 30200195). We thank Dr Hai-Bin Wang for taking photographs and Dr Su-Hui Wu (Henan Normal University, China) for statistical analysis. We thank the faculty of Huanghuai University for supporting Dr En-Zhong Li.

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