Testicular descent is an important aspect of male sexual development. Cryptorchidism is the failure of the testis to descend into the scrotum, affecting approximately 3% of full-term male infants. It can result in infertility and is associated with an increased risk for testicular torsion and development of testicular cancer.1 Normal testicular descent has been described as two phases, the transabdominal phase and the inguinoscrotal phase.2 During the transadominal phase, the testis migrates from the urogenital ridge to the inguinal region, which occurs from 10-15 weeks in the embryo of human and from embryonic day 15.5 (E15.5) to E17.5 in the rat.3
It has been widely accepted that the increased presence in the environment of xenoestrogens in the past 50 years, may be the cause for declining male reproductive health, such as the increased incidence of cryptorchidism.4 Diaethylstilbestrol (DES) was commonly used in the 1950s and 1960s to lower the risk of spontaneous abortion. Currently, it is associated with undescended testis regarded as a classic xenoestrogen.5,6 Numerous reports also showed that transabdominal testicular descent abnormalities in rodent fetuses are induced by prenatal exposure to DES.7,8 The mechanism of DIIAC is not completely clear. Considering the importance of the INSL3/LGR8 system9-11 and HOXA1012,13 in transadominal testicular descent, we investigated their roles in DIIAC. Additionally, we also investigated the effect of DES on SF-1, which has been reported to control transcription of INSL3.14
Fifty female Sprague-Dawley rats (200-220 g, purchased from the Center of Animal, Medical School of Wuhan University) were mated and assigned to five groups. The presence of the vaginal plug was counted as day E0.5. Pregnant rats received a subcutaneous injection of 0, 2.5, 5, 10, and 20 mg/kg DES (Sigma, USA) at day E13.5 and were referred to as groups A, B, C, D, and E, respectively. The rats in group A were administered only DMSO as a control. The time of injection was selected as described previously.7 At E19.5, all rats underwent a Caesarean section and male fetal mortality was determined. Male fetuses from five rats of each group were fixed overnight in 4% paraformaldehyde for determining degree of transabdominal testicular ascent (DTA). Male fetuses from another five rats of each group were killed by decapitation, their testes and gubernaculums were removed quickly using an operating microscope, and then stored in liquid nitrogen until RNA was isolated. In order to correctly perform Caesarean section in rats and remove fetal gubernaculums accurately, we have been trained by postgraduate in obstetrics who is experienced with Caesarean sections in rats, and by an experienced microsurgeon. After we acquired the techniques all the operations were performed by the same operators.
To investigate if DMSO could affect transabdominal testicular descent and fetal mortality, another ten pregnant Sprague-Dawley rats were separated into two groups and received a subcutaneous injection of 1ml normal saline (NS) or 1ml DMSO at E13.5 and were named group NS or group DMSO. The protocol for determining DTA and fetal mortality of these two groups is identical with that of groups A through E.
After the abdominal wall was removed and the abdominal cavity cleared, DTA was determined using a stereomicroscope (Zeiss, Germany) by measuring the distance from the bladder neck to the lower pole of the testis, as described previously.15 The measurements were standardized by defining the distance between the bladder neck and the lower pole of the kidney as 100 units.
Semiquantitative reverse transcriptase PCR
Total RNA of testes and gubernaculums were extracted by Trizol reagent (Gibco, USA) and quantified by spectrophotometry. The mRNA was reverse-transcribed to cDNA in a total volume of 20 μl with random primers (Promega, USA) using M-MLV reverse transcriptase (Promega, USA) at 25°C for 10 minutes, 42°C for 60 minutes, and 93°C for 3 minutes. The PCR program consisted of an initial denaturation at 94°C for 5 minutes, followed by 35 cycles of denaturation at 94°C for 40 seconds, annealing for 60 seconds, elongation at 72°C for 60 seconds, and a final elongation at 72°C for 7 minutes. β-actin was amplified simultaneously as internal control. The primers, PCR products, and annealing temperatures of each gene are listed in Table 1.
After extraction of testis protein, protein concentration was measured by ultraviolet spectrophotometer (Biochrom Ltd, England). Subsequently, 20 μg of total protein from each sample was loaded on 15% SDS-PAGE. For Western blotting analysis, gels were electroblotted to a nitrocellulose membranes, which were then blocked for 1 hour and incubated for 1 hour at room temperature in the presence of INSL3 antibody (Boster, China, diluted 1:800). The membranes were washed in TBST then incubated for 1 hour at room temperature with peroxidase-conjugated goat anti-rabbit IgG (PIERCE, USA, diluted 1:3000). Bound antibodies were detected using an ECL plus system and Hyperfilm exposure (Amersham Biosciences, UK). GAPDH protein was used as an internal control.
Statistical analysis was performed using SPSS 11.0 software. The results were analyzed using an analysis of variance (ANOVA) test. All data in this paper are presented as the mean ± standard error of the mean (SEM), with P <0.05 described as statistical significance. All bands from RT-PCR and Western blotting were semiquantified with Quantity One software.
Fetal mortality and DTA
Each DES-treated group showed vaginal bleeding, which was more severe as the dose increased. There was one absorbed fetus in group D and four in group E. The fetal mortality in groups A, B, C, D, E were 3.57%, 6.90%, 12.00%, 19.23% and 36.36%, respectively. Fetal mortality in the DES-treated groups was significantly higher than group A and increased in a dose-dependent manner (P <0.01, Table 2). As shown in Figure 1, the testes of rats in group A were localized close to the inguinal region and near the bladder neck, whereas the testes of the DES-treated groups were localized above the bladder neck or, in some cases, just below the kidney. The DTA of the DES-treated groups was higher than that of group A and increased in a dose-dependent manner (P <0.01, Table 2).
There was no significant difference in fetal mortality between group NS (4.00%, 1/25) and group DMSO (4.17%, 1/24) (P >0.05), nor in DTA between group NS (8.2%±1.4%) and group DMSO (8.1%±1.5%, P >0.05, Figure 2). These results indicate that DMSO had no effect on transabdominal testicular descent and fetal mortality.
mRNA expression of INSL3, SF-1, and HOXA10 and protein expression of INSL3
Compared with group A, the INSL3 mRNA in DES-treated groups decreased significantly in a dose-dependent manner (Figure 3P <0.01), so were the INSL3 protein (Figure 4) and SF-1 mRNA. Moreover, the expression of INSL3 mRNA and protein in group E almost could not be detected. Compared with group A, LGR8 mRNA in group B and C was not significantly different (P >0.05), but those in group D and group E increased significantly (P <0.05, P <0.01, respectively). There was no significant difference about HOXA10 mRNA between group A and group B, but those in groups C, D and E decreased significantly (P <0.05, P <0.01, P <0.01, respectively, Table 3).
In this study, we find that the gubernaculum in the rats is difficult to remove exactly because of its small size and the adhesion by adjoining tissues, which may affect the investigation on gubernaculums. Exact removal of the gubernaculum in the rat is easier to perform using an operation microscope, which makes it possible to investigate the LGR8 and HOXA10 mRNA in gubernaculums in DIIAC.
As intra-abdominal testicular descent occurs from E15.5 to E17.5 in rats, we tried daily subcutaneous injecting different doses of DES into pregnant females from E15.5 to E17.5 for establishing DIIAC model in rats in our preliminary experiment, but failed to get a stable model (data not shown). Intra-abdominal cryptorchidism in mice has been induced by subcutaneous injecting 17β-estradiol into pregnant females at E13.5.7 In order to realize it in rats, we attempted to administrate DES into pregnant rats subcutaneously at E13.5 at a dose of 0, 2.5, 5, 10, 20 mg/kg, respectively. Results indicate that 20 mg/kg of DES leads to satisfied DTA but too high fetal mortality (36.36%), 2.5 and 5 mg/kg of DES are satisfied in fetal mortality but not in DTA. Therefore, we suggest that it may be an acceptable method for establishing DIIAC model in rats to administrate DES (10 mg/kg) into pregnant rats subcutaneously at E13.5. INSL3 transcripts are first detected in murine testis at E13.5,16 injection at E13.5 is conducive to investigating the role of INSL3 in DIIAC. A single injection is also easy to perform. However, multiple low-dose exposure to DES in fetal rats is more similar to DIIAC in humans than a single dose, thus further study on DIIAC model in rats should focus on multiple low-dose exposure to DES in key stage of transabdominal testicular descent. In addition, results of present study confirm that prenatal exposure to DES in the rat inhibits transabdominal testicular descent dose-dependently, which is consistent with previous studies.7,8
Mice mutants for INSL3 exhibit bilateral cryptorchidism and developmental abnormalities of the gubernaculum, but no obvious defects to the male reproductive organs.9,10 These establish a crucial role of INSL3 in transabdominal testicular descent and gubernacular growth. Furthermore, INSL3 has been proved to have a direct stimulatory effect on fetal gubernacular growth.17 In present experiment, DES inhibited transabdomial testicular descent dose-dependently. Simultaneously, DES decreased the expression of INSL3 mRNA and protein dose-dependently. These results confirm that prenatal exposure to DES in the rat leads to transabdomial testicular maldescent via decreasing the expression of INSL3 mRNA and protein dose-dependently.
The mechanism that DES down-regulates the expression of INSL3 is still not clearly, and remains controversial. As a transcription factor,18 SF-1 can increase the INSL3 expression by direct binding to the specific sites within the INSL3 promoter, has been reported to control transcription of INSL3.14 Nef et al7 and Emmen et al8 found that DES treatment had no effect on SF-1 expression, whereas Majdic et al19 observed a decrease in SF-1 mRNA in testes of DES-exposed fetuses. Our results show that DES down-regulates SF-1 mRNA expression in fetal testis dose-dependently, so we suggest that DES down-regulates INSL3 expression via down-regulating SF-1 in testes of DES-exposed fetuses. Moreover, RT-PCR showed that the expression of INSL3 almost can not be detected in group E and the expression of SF-1 in the same group is still detectable. Therefore, we speculate that other factors may be involved in decreased INSL3 by DES besides SF-1. Additionally, it is interesting to point out that the animals are rats in all the studies in which SF-1 was down-regulated by DES,19 including the present study, whereas the animals are mice in the studies in which SF-1 was not affected.7,8 With respect to the effect of DES on SF-1, are there any differences between rats and mice? If positive, further studies on the mechanism are needed.
LGR8 is the only receptor for INSL3,20 and the LGR8 receptor is high expressed in the gubernaculums.17 GREAT knockout mice have an identical cryptorchid phenotype to that of INSL3 knockout mice.11 However, it remains to be determined whether or not LGR8 is involved in DES-induced transabdominal testicular maldescent. LGR8 mRNA was not affected at 2.5 mg/kg and 5 mg/kg of DES, and increased at 10 mg/kg and 20 mg/kg of DES. As not down-regulated, LGR8 may not be responsible for DES-induced transabdiminal testicular maldescent. It is unknown that the increase of LGR8 mRNA was induced by DES or compensated after the decrease of its ligand, INSL3. Examining the change of the LGR8 expression in gubernaculums treated by DES in vitro may be useful for this issue. The mechanism that DES up-regulates LGR8 is still unknown. During the preparation of this manuscript, Feng et al21 reported that the Sry-related high mobility group box 9 (SOX9) might play a specific role in regulating LGR8 activity. This could offer a way to explore the mechanism.
HOXA10 is only predominantly expressed in the gubernaculum and kidney, not in other organs and tissues of male reproductive system.13 HOXA10 knockout mice exhibit unilateral or bilateral intra-abdominal cryptorchidism, with normal androgen production and abnormalities of the gubernaculum as well as abnormalities of vertebrae and lumbar spinal nerves.12,13 These imply that HOXA10 is also an important gene for transabdominal testicular descent and gubernacular growth. It also remains to be determined that if HOXA10 is involved in DES-induced transabdominal testicular maldescent. In the present study, HOXA10 mRNA was not affected at 2.5 mg/kg of DES, and decreased at 5, 10, and 20 mg/kg of DES. These results imply that HOXA10 is not involved in DES (2.5 mg/kg) induced transabdiminal testicular maldescent, but involved in those by DES (5, 10, and 20 mg/kg). These results also indicate that a direct action of DES upon the gubernaculum could exist. Furthermore, we think over a question. Which one is predominant between INSL3 and HOXA10 in DIIAC? It seems that INSL3 is predominant in that by DES (2.5 mg/kg), because INSL3 was down-regulated in that by DES (2.5 mg/kg) but HOXA10 was not affected. In those by DES (5, 10, and 20 mg/kg), both INSL3 and HOXA10 were down-regulated and more significantly in INSL3, but it should be prudent to conclude that INSL3 is predominant. Besides, abnormalities of vertebrae and lumbar spinal nerves were not observed in our study, perhaps it is because HOXA10 is not low enough to induce these abnormalities.
In conclusion, DES can inhibit transabdominal testicular descent dose-dependently via dose-dependent down-regulating the expression of INSL3, which was induced by dose-dependent down-regulating the expression of SF-1. HOXA10 may be not involved in DES (2.5 mg/kg) induced abdominal cryptorchidism, but involved in those by DES (5, 10, and 20 mg/kg). LGR8 may be not responsible for DES-induced transabdominal testicular maldescent.
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