Association of testicular undescent induced by prenatal flutamide treatment with thickening of the cremaster muscle in rats

Abstract

Background and Aims:  Previously, in cryptorchid rats, which were induced by prenatal exposure to flutamide, we found a thickening of the cremaster muscle. This study was undertaken to quantify the increase of the cremaster muscle thickness in the cryptorchid rats, and to examine its possible relationship with the proliferation of muscle cells.

Methods:  To obtain cryptorchid rats, pregnant Wistar rats were subcutaneously injected with flutamide (100 mg/kg per day) during gestational days 16–17. Serial sections of the scrotum, containing the testis and cremaster muscle, were prepared from the control and cryptorchid rats that were 2–6 weeks of age, and stained with hematoxylin–eosin for morphometry, or stained with antibody against the proliferating cell nuclear antigen (PCNA) to analyze the cell proliferation ability.

Results:  The thickened cremaster muscle was always associated with cryptorchid testis and, in the case of unilateral cryptorchidism, the cremaster muscle of the contralateral (descended testis) side exhibited normal thickness. The average thickness of the affected cremaster muscle was 0.80 and 1.89 mm at 4 and 6 weeks of age, respectively, although that of the normal muscle was 0.28 and 0.33 mm at the same time period, respectively.

Conclusion:  Our results showed that the cremaster muscle of the cryptorchid rats was significantly thicker than that of the control rats. The immunohistochemical analysis revealed that a thickened cremaster muscle contained many PCNA‐positive nuclei even at 4 weeks of age, in contrast to the control, which had only a few positive nuclei. Our present study indicates that continuous proliferation of the muscle cells associated with cryptorchid testis increases the thickness of cremaster cells in rats exposed to flutamide prenatally. (Reprod Med Biol 2003; 2: 109–113)

INTRODUCTION

Testicular descent is a complex process in which two phases of hormonal controls have been hypothesized. ¹ , ² During the first phase, or transabdominal descent, the testis move from its original position near the kidney to the bottom of the abdomen, and reaches the bladder neck by birth. This phase has been thought to be controlled by the Müllerian inhibitory factor (or substance), but this is under controversy. ³ The second phase (inguinoscrotal or transinguinal descent) is the androgen‐dependent descent of the testis from the internal inguinal ring to the scrotum.

Flutamide, a non‐steroidal androgen antagonist, has been known to block the transinguinal descent of the testis when it was prenatally administrated to the fetus. ⁴ , ⁵ , ⁶ , ⁷ , ⁸ , ⁹ In our previous study of the rat cryptorchidism using flutamide, ¹⁰ we encountered thickened cremaster muscles in cryptorchid rats. The thickening of the cremaster muscle has been reported to occur in estrogen receptor knockout (ERKO) mice. ¹¹ To the best of our knowledge, however, there is no detailed report describing the association of thickening of the cremaster muscle with testicular undescent induced by flutamide.

The aim of this study was to quantify the increase of the cremaster muscle thickness in cryptorchid rats, which is induced by prenatal flutamide treatment, and also to examine its possible relationship with the proliferation of muscle cells.

MATERIALS AND METHODS

Animals

Wistar rats were obtained from Japan SLC Inc. (Hamamatsu, Japan). The rats were housed under standard animal housing conditions in accordance with the Chiba University Guide for the Care and Use of Laboratory Animals. Each male rat was placed in individual cages with two female rats for mating purposes. Gestational day 0 (G0) was designated when a vaginal plug was found.

To obtain cryptorchid rats, a total of 36 pregnant rats were subcutaneously injected once in a day from G16 to G17 with flutamide (100 mg/kg per day; Sigma‐Aldrich, Tokyo, Japan) solved in 0.5 mL of 20% ethanol. ⁵ In the rat offspring, a total of 57.7% (104 of 180) of testes were found to be located close to the neck of the bladder or at an upper position of the scrotum. Six, 10 and eight male rat offspring were killed at 2, 4 and 6 weeks of age, respectively, and used for the experiments. For the control, five pregnant rats were subcutaneously injected once in a day from G16 to G17 with 0.5 mL of 20% ethanol. The male rat offspring of the same number as cryptorchid rats were used. The abdominal and scrotal skins were excised, and the testicular location was anatomically verified under a low power microscope.

Collection and treatment of tissue

The scrotum, containing the testis and cremaster muscle, was removed from the cryptorchid and control rats when they were 2, 4 and 6 weeks of age, and fixed in 4% paraformaldehyde in 0.1 mol/L phosphate buffer (pH 7.5) at 4°C for 16 h. The tissues were dehydrated with the use of graded alcohols, and embedded in paraffin. Serial sections were cut at 7 µm in a microtome, attached to slides coated with 3‐aminopropyltriethoxy silane (Matsunami Glass Ind., Ltd, Osaka, Japan), deparaffinized with xylene and ethanol series, and then used for hematoxylin–eosin and immunohistochemical stainings.

Immunohistochemical staining

To analyze the proliferation ability of the cells, the sections were stained with mouse monoclonal antibody against the proliferating cell nuclear antigen (PCNA; Medical & Biological Laboratories, Co., Nagoya, Japan). These sections were treated with biotinylated antimouse immunoglobulin (Ig)G followed by peroxidase H linked with avidin (VECTASTAIN Elite ABC kit; Vector Laboratories Inc., Burlington, CA, USA). They were then treated with diaminobenzidine and H₂O₂ at 25°C for 20 min so that immunoreactive nuclei could be visualized.

Observation and quantitative analyses

The specimens were examined with the use of a microscope, Zeiss Axioskope 2 plus, equipped with a scanning digital camera AxioCam (Carl Zwiss Co., Baden‐Württemberg, Oberkochen, Germany). For analysis of the cremaster muscle thickness, sections in which the scrotal wall was cut rectangular to its surface were selected. In the sections, the maximum thickness of the cremaster muscle layer was measured at the point near the bottom of the scrotum. Raw data of the cryptorchid group were collected from 10 and eight unilateral cryptorchid rats at 4 and 6 weeks, respectively, and the mean ± SD were calculated. The raw data of the control group were also collected from the same number of control rats, and treated in the same way as the cryptorchid group. Significant differences between the cryptorchidism and control groups were analyzed by the following statistical methods. The homogeneity of variances for the parameters of thickness of the cremaster muscle was analyzed by using the F‐test. When a set of variances was not homogenous, the parameter was analyzed by using the Welch T‐test.

For quantification of anti‐PCNA positive nuclei, labeled nuclei were counted in four optical areas of the cremaster muscle in each section (n > 300). The anti‐PCNA labeling index was calculated as the number of labeled nuclei/number of total nuclei × 100 (%). The indication of immunopositive nuclei was scored as follows: –, no staining (0%); ±, very low level (<5%); +, moderate level (5–20%); ++, significant level (>20%).

RESULTS

Morphology of the cremaster muscle in cryptorchid rats

Figure 1 shows a frontal section of the pubic region of a typical cryptorchid rat at 6 weeks of age stained with hematoxylin–eosin. ¹⁰ In this rat, the left testis was descended into the scrotum but the right testis was undescended (Fig. 1a). The cremaster muscle lining the right scrotum was much thicker than that of the left side (Fig. 1a). At a higher magnification, the cremaster muscle of the undescended side showed many foldings (Fig. 1b). The remnants of the gubernacular bulb (GB) and the gubernacular cord (GC) were found near the bottom of the right scrotum (Fig. 1b). However, such remnants were not observed in the left scrotum.

Figure 1.

Frontal section of the pubic region of a cryptorchid rat aged 6 weeks stained with hematoxylin and eosin. ¹⁰ (a) Cryptorchidism occurred on the right testis (RT), but the left testis (LT) was descended into the scrotum. The right testis was smaller than the left testis, and the right cremaster muscle (CM) was thicker than the left one. (b) Higher magnification of the region indicated by a rectangle in (a). The remnants of the gubernacular bulb (GB) and gubernacular cord (GC) were detected. E, epididymis; P, penis. Bar, 2 mm.

Thickness of the cremaster muscle

The thickness of the cremaster muscle was measured in the control and cryptorchid rats at 4 and 6 weeks of age (Fig. 2; Table 1). In the control rats, the cremaster muscle was well extended and its surface facing the scrotal lumen was very smooth (Fig. 2a,c). Average thickness of the cremaster muscle of normal rats was 0.28 and 0.33 mm at 4 and 6 weeks of age, respectively (Table 1). In contrast, on the undescended side of the cryptorchid rats, the cremaster muscle exhibited many foldings at the luminal surface, and was obviously thicker than that of the normal rats (Fig. 2b,d). The average thickness of such muscle layer of cryptorchid rats was 0.80 and 1.89 mm at 4 and 6 weeks of age, respectively (Table 1). Thus, the cremaster muscle on the cryptorchid side exhibited a three‐ and fivefold increase in thickness as compared with the control muscle at 4 and 6 weeks of age, respectively.

Figure 2.

Cremaster muscle (CM) near the bottom of the scrotum stained with hematoxylin and eosin. (a) Control rat aged 4 weeks. (b) Cryptorchid rat aged 4 weeks (testis‐undescent side). (c) Control rat aged 6 weeks. (d) Cryptorchid rat aged 6 weeks (testis‐undescent side). The cremaster muscles of cryptorchid rats (b, d) were thicker than those of control rats (a, c). T, testis. Bar, 1 mm.

Table 1.

Thickness of the cremaster muscle in rats

Age (weeks)	n	Control	Cryptorchidism
4	10	0.28 ± 0.07	0.80 ± 0.18*
6	8	0.33 ± 0.03	1.89 ± 0.07*

Data shows mean (mm) ± SD. *Significant difference from control (P < 0.001). n, no. samples.

The histology of the cremaster muscles showed normal muscle fibers both in the control (Fig. 2a,c) and cryptorchid rats (Fig. 2b,d). The interstitial spaces among muscle fibers (perimysium and endomysium) seemed to show no differences between the normal and thickened cremaster muscles.

Cell proliferation in the cremaster muscle

The proliferation ability of the cremaster muscle cells was investigated by staining with anti‐PCNA when the rats were 2 and 4 weeks of age (Fig. 3; Table 2). In the control cremaster muscles, approximately 10% nuclei were positive for PCNA at 2 weeks of age (Fig. 3a), but only several nuclei were labeled with anti‐PCNA at 4 weeks (Fig. 3c). Thus, in the normal cremaster muscle, the cell proliferation ability decreased in accordance with the growth of the muscle (Table 2). In the cryptorchid rats, the cremaster muscle of the undescended side contained many PCNA‐positive cells (Fig. 3b,d), and the labeling indices were more than 20% at both 2 and 4 weeks of age (Table 2).

Figure 3.

Cremaster muscle near the bottom of the scrotum stained with antibody against proliferating cell nuclear antigen (PCNA). (a) Control rat aged 2 weeks. (b) Cryptorchid rat aged 2 weeks (testis‐undescent side). (c) Control rat aged 4 weeks. (d) Cryptorchid rat aged 4 weeks (testis‐undescent side). In cremaster muscles of control rats, approximately 10% nuclei were labeled with anti‐PCNA at 2 weeks of age (arrow in a), but only few nuclei were positive for PCNA at 4 weeks (c). In cremaster muscles of cryptorchid rats, however, more than 20% of nuclei were PCNA‐positive at both 2 and 4 weeks of age (arrow in b and d, respectively). Bar, 1 mm.

Table 2.

Immunoreactivity of the cremaster muscle for anti‐proliferating cell nuclear antigen

Age (weeks)	n	Control	Cryptorchidism
2	6	+	++
4	6	±	++

Labeling index was calculated as (number of labeled nuclei/number of total nuclei) × 100%, and the immunoreactivity was scored as follows: –, no staining (0%); ±, very low level (<5%); +, moderate level (5–20%); ++, significant level (>20%). n, no. samples.

DISCUSSION

In the control rats, the migration of testes into the scrotum was completed by 4 weeks of age. Both GB and GC were detectable at 2 weeks after birth (data not shown), but became undetectable in the control rats when they were 4 weeks old. These results are consistent with a report describing normal testicular descent. ¹² Several investigators have reported that prenatal exposure to flutamide or other chemicals induced cryptorchidism. ⁴ , ⁵ , ⁶ , ⁷ , ⁸ , ⁹ , ¹³ In the cryptorchid rats induced by prenatal exposure to flutamide, GB and GC were undetectable on the side of the descended testis at 4 weeks of age, but they detectable on the side of the undescended testis. This suggests that regression of the gubernaculum, as well as testicular descent, can normally occur even in the cryptorchidism induced by prenatal exposure to flutamide.

The thickening of the cremaster muscle has been noted in ERKO mice. ¹¹ As shown in the present study, also in prenatally flutamide‐treated rats, the thickened cremaster muscles were found in the cryptorchid rats. However, in contrast to the ERKO mice, the thickening of the cremaster muscle was always associated with undescended testis of unilateral cryptorchidism, and the cremaster muscle of the contralateral side where the testis was descended into the scrotum exhibited normal morphology. This indicates that the thickening of the cremaster muscle is related to the occurrence of testis undescent rather than the direct effect of flutamide.

The immunohistochemical analysis using anti‐PCNA revealed that the thickened cremaster muscle contained many PCNA‐positive nuclei at both 2 and 4 weeks of age, although PCNA‐positive nuclei decreased in number in the normal cremaster muscles by 4 weeks. This suggests that the increase of the cremaster muscle thickness is caused by the proliferation of myoblasts because we could not find any increase in areas of perimysium and endomysium in the thickened cremaster muscle. The present results also suggest that the differentiation of the thickened cremaster muscle is delayed. It has been well known that differentiated muscle cells lack proliferation ability. Moreover, it has been found that the cremaster muscles associated with undescended testes contain embryonic isoforms of muscle proteins. ¹⁰ These facts support the present idea.

In the present study, we can not conclude whether the thickening of the cremaster muscle is the cause or the resultant of the testicular undescent. As suggested in the ERKO mice, however, there is a possibility that the thickened cremaster muscle may retract the testis more easily into the abdomen than unaffected muscle would.

In conclusion, it has been shown that the cremaster muscle of the cryptorchid rats was significantly thicker than that of the control rats. It was also indicated that continuous proliferation of the muscle cells associated with the cryptorchid testis, which increased the thickness of the cremaster cells in rats exposed to flutamide prenatally. Further studies are required in order to address such issues.