ISI Impact Factor (2004): 1.096

Editor-in-Chief
Prof. Yi-Fei WANG,

Effect of prenatal exposure to diethylstilbestrol on gubernacular development in fetal male mice

Xue-Wu Jiang¹, Jian-Hong Li², Tian-Hua Huang², Wang-Dong Deng¹

¹Department of Pediatric Surgery, Second Affiliated Hospital of Shantou University Medical College (SUMC), ²Research Center for Reproductive Medicine, SUMC, Shantou 515041, China

Asian J Androl  2004 Dec; 6: 325-329         

Keywords: diethylstilbestrol; estrogen; gubernaculum

Abstract

Aim: To study the effect of prenatal exposure to diethylstilbestrol (DES) and the role of actin and proliferating cell nuclear antigen (PCNA) on testicular gubernaculum development in fetal male Kunming mice. Methods: Pregnant mice were randomly assigned to 6 groups and injected with DES subcutaneously from gestational day 9 (E9) to day 17 (E17) at doses of 0, 25, 50, 100, 200 µg·kg^-1·d^-1 in 0.2 mL dimethyl sulfoxide (DMSO). On E17 they were sacrificed and fetuses quickly removed for fixation. Male fetuses were sliced on serial coronal plane. Histological changes were observed under the light microscope (LM) and ultrastructural changes with the scanning and transmission electron microscopes (SEM & TEM). The expression intensity of actin and PCNA in the gubernacula was quantitated by immunohistochemistry. Results: The mortality of the fetuses was higher in the DES-treated groups than that in the DMSO and saline controls (P<0.05). Under LM the gubernacula were seen to be poorly developed with smaller bulbs. On SEM the bulbs lose the clear demarcation between the mesenchymal inner core and the muscular outer layer and looked like a small cone instead of the normal cylindrical appearance. On TEM there were some smaller disordered myofibrils and sparse cytoplasmic organelles in the gubernacular muscular cells of the treated groups. The expression intensity of actin and PCNA in the gubernacula was significantly weaker in the treated groups than that in the DMSO and saline controls (P<0.05). Conclusion: DES induces underdevelopment of the gubernacula in a dose-dependent manner in fetal male mice and down regulates the actin and PCNA expression.

1 Introduction

Recently, there is a great concern on the increasing incidence of congenital disorders of male sexual differentiation, cryptorchidism, hypospadia, micropenis, etc [1- 4]. For example, in England the incidence of cryptorchidism has doubled within the last 25 years [5]. It has been suggested that they may be related to environmental xenoestrogens and/or antiandrogens.

In mice, rats and hamsters, exposure to elevated level of estrogens increased the incidence of cryptorchidism[6, 7]. It was proposed that estrogens disturbed the fetal hypothalamus-pituitary axis and blocked gonadotropin secretion and consequently, inhibited the androgen production. It has also been suggested that estrogens might have a direct effect on the gubernacula [8-10]. Nef et al [11] indicated that estrogen damaged the gubernacular morphology and impeded testicular descent.Donaldson et al [6] reported the over-development of cremaster muscle associated with testicular retraction in an estrogen receptor knockout (ERKO) mouse. It could be seen that estrogen played an important role in guber-nacular development and testicular descent. However, which components of the gubernacula and how they were affected remained unclear. The effects of prenatal exposure to diethylstilbestrol (DES) provide a satisfactory model of how a potent estrogenic chemical prescribed during gestation can alter the fetal sex differentiation [12]. The effects of DES on the gubernacular development have seldom been reported and thus were investigated in the present study.

2 Materials and methods

Female Kuming mice, 32 ± 3 g, were provided by the Animal Care Center of this University. After mating, vaginal smear was performed every morning and the observation of a positive smear was considered the gestational day 0 (E0). Pregnant mice were randomly assigned to 6 groups of 10 mice each and injected DES (Sigma, USA) subcutaneously from day 9 (E9) to day 17 (E17) at doses of 0, 25, 50, 100, 200 µg·kg^-1·d^-1 in 0.2 mL dimethyl sulfoxide (DMSO, Amresco, USA), respectively. Another group was injected the same volume of normal saline (NS) alone.

Pregnant mice were sacrificed on E17 and fetuses were quickly removed by cesarean section. The male fetuses were fixed in 10 % neutral formaldehyde for 24 hours, embedded in paraffin and sliced (4 µg) on serial coronal plane. The sections were stained with hemyotoxin-eosin (HE) for immunohistochemistry study. For scanning electron microscopy (SEM), male fetuses were fixed in 2.5 % glutaraldehyde, osmicated, dehydrated and embedded in epoxy-resin. For transmission electron microscopy (TEM), ultrathin sections were stained with uranyl acetate and lead citrate, and examined with a Hitachi-600 electron microscope operated at 60 Kv (Hita-chi, Japan).

After fixation in 10 % neutral formaldehyde, 4 mm thin coronal sections were cut and mounted on polylysine-coated glass slides. Immunohistochemistry was performed using the streptavidin-biotin-avidin complex (SABC) method. A rabbit antimouse actin (muscle type) and proliferating cellular nuclear antigen (PCNA) polyclonal antibodies (Boster Co., China) at 1:100 dilutions were used as the primary antibody. Biotin-labeled goat anti-rabbit (Boster) was used as the secondary antibody at a 1:200 dilution. Phorsphoric acid buffer solution (PBS) instead of actin and PCNA polyclonal antibodies was used as controls. Visualization of brown granules in the cytoplasm was judged as positive for actin expression and yellow-brown nuclei for PCNA. The expression intensity was quantitated by computer analysis in terms of their grey scales. The deeper the color and the more the number of the granules, the stronger the expression intensity of the tissue is.

Data were presented as mean ± SE. The results of actin and PCNA were compared using the Student's t-test. P<0.05 was considered significant.

For animals treated with DMSO, NS and 25, 50, 100, 200 µg·kg^-1·d^-1 of DES, the fetal mortality was 2.15 %, 3.30 %, 6.67 %, 11.63 %, 18.37 % and 31.82 %, respectively. The larger the doses of DES, the higher the fetal mortality would be, suggesting a dose-dependent effect. The mortality of the fetuses was significantly higher in the DES-treated groups than that in the DMSO or NS groups (P < 0.05).

The gubernacula of DMSO and NS groups were fully developed and consisted of two parts: gubernaculum bulb and cord. The former was well developed with well-demarcated mesenchymal inner core and muscular outerlayer (Figure 1A, B). The muscular cells were dense and well arranged in bundle. On the contrary, the gubernacula in the DES-treated fetuses seemed to be undifferentiated with a relatively smaller bulb volume (Figure 1C, D). The demarcation between the mesenchymal core and the muscular outerlayer was unclear and the muscular cells, sparsely scattered. As the dose of DES was increased the histological structure became more and more disordered.

Figure 1. Histology of fetal mice on E17 revealed the gubernacula of normal and DES-treated groups.
(A) NS (normal) group. Well-developed gubernaculum bulb (GB), with clear demarcation between the core and outlayer was adhered with lower peritoneal wall (LPW) (×40) .
(B) Illustration of normal bulb: inner mesenchymal core and muscular outerlayer was clearly demarcated (×400).
(C) DES 25 µg·kg^-1·d^-1 group. Nearly normal bulb, similar to (A) (×40).
(D) DES 200 µg·kg^-1·d^-1 group. Much smaller bulb with unclear demarcation between the core and outlayer (×40).

In normal fetus, SEM observation indicated that the well-developed bulbs appeared to be a cylinder with a normal volume and the cords, stout and short (Figure 2A). The testes were fully developed and descended to the lower abdomen near the bladder (complete testicular descent). By contrast, in the DES-treated fetuses, the poorly developed bulb looked like a small cone and appeared flat and thin with a smaller volume and the cords were elongated and slim (Figure 2B). The poorly developed testes were located at the lower pole of the kidney (incomplete testicular descent).

On TEM, the gubernacular cells of the normal group were well-arranged with bigger myofibrils, the electron-dense bodies were obvious with many filaments (Figure 3A). There were abundant organelles in the cytoplasm, suggestive of active cellular metabolism. The gubernacular cells of the DES-treated groups had smaller and disordered myofibrils with few filaments (Figure 3B) and only some sparse organelles were seen scattered in the cytoplasm .

The expression intensity of the sections was analyzed in terms of their grey scales and the deeper color of each cell and/or the increase in the number of actin- or PCNA-positive cells imply a higher scale value and a stronger expression intensity. The results are shown in Table 1 and Figure 4. The expression of actin and PCNA in the gubernaculum was significantly weaker in the DES-treated groups than that in the DMSO and normal groups (P<0.05). The bigger the DES dose, the weaker the expression intensity would be.

Table 1. Expression intensity of actin and PCNA in different groups. ^bP < 0.05, compared with NS.

Figure 4. Immunohistochemistry. (A) Actin in DMSO group. Strong positive granules were distributed in the cytoplasm of muscular outlayer. Similar for NS group. (B) In DES 50 µg·kg^-1·d^-1 actin was weakly positive. (C) PCNA in NS group. Yellow-brown nuclei suggested positive for PCNA. (D) PCNA in DES 200 µg·kg^-1·d^-1. PCNA-positive cells were decreased. (×400)

4 Discussion

The testicular gubernaculum played an important role in testicular descent, which was closely related with testicular development. However, the effect of estrogen on the gubernacular morphology and development has seldom been studied.

In the present study, after different doses of DES the flat and thin bulbs and the elongated and slim cords were clearly demonstrated both on LM and SEM. In combination with the smaller disordered myofibrils on the TEM, all the findings confirmed that DES could directly influence the morphological development of the gubernacula and the decent of testis, which was consistent to the observation of Shono et al [13]. The present observation clearly showed that the morphological changes were graded in a dose-dependent manner.

The mesenchymal cells in the bulbs actively proliferate and differentiate into the cremasteric cells and during this process PCNA acts as an index of the degree of cell proliferation [14, 15]. The expression intensity of both actin and PCNA in the gubernacula was significantly weaker in the DES-treated than in the DMSO and normal groups. The bigger the dose of DES, the weaker would be the expression intensity. Reduced expression of the actin lead to reduced contractility of the thin filament; down-regulation of PCNA suggested the inhibition of proliferation activity of the gubernacular cells.

As to the mechanism of DES effect on the gubernacular components, recent insight came from the studies on the insulin-3 (insl3) gene [7, 10] and it was indicated that DES might cause a specific down-regulation of the Insl3 transcription and expression, suggesting a role of insl3 gene in estrogen-induced cryptorchidism.

In conclusion, DES not only induces gubernacular underdevelopment in a dose-dependent manner, but also down-regulates the expression of actin and PCNA in the gubernacula, which at least partially contributed to the decline of its contraction activity, and inhibits gubernacular cell proliferation and consequently, testicular decent and development.

The work was supported by the National Nature Science Fundation of China (No. 30170481) and the Science and Technology Program Fundation of Guangdong Province (No. C31302).

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Correspondence to: Dr. Tian-Hua Huang , Research Center of Reproductive Medicine, SUMC, Shantou 515041, China.
Tel: +86-754-890 0442, Fax: +86-754-855 7562
Email: thhuang@stu.edu.cn
Received 2003-11-13   Accepted 2004-06-02