The crossbreeding of the August 1561 and Copenhagen 2331 lines in 1926 created the ACI rat strain. Following the first 16 generations of continuous brother–sister inbreeding, it was noted that the ACI rat was prone to the development of urogenital abnormalities (Morgan, 1953; Deringer and Heston, 1956). Numerous studies over the past 50 years have confirmed the observation of these abnormalities with 10%–25% of the animals affected predominantly on the right side (Deringer and Heston, 1956; Fujikura, 1970; Cramer and Gill, 1975; Maekawa and Odashima, 1975; Shoji and Harata, 1977; Marshall et al., 1978; Fujita et al., 1979; Marshall et al., 1979; Marshall et al., 1982; McCullough et al., 1984; Solleveld and Boorman, 1986; Kneidl et al., 1995; Shull et al., 2006). The urogenital abnormalities include ipsilateral hypoplasia (IHP) of the epididymis (ED), vas deferens (VD), and seminal vesicle (SV) in males; the presence of a single uterine horn, and in some cases, a single ovary in females; and the ipsilateral absence or truncation of the ureter in both sexes (Deringer and Heston, 1956; Marshall et al., 1978). More severe defects of the urogenital tract have also been described, including hydronephrosis or the complete absence of a kidney ipsilateral to the urogenital agenesis in both sexes (Marshall et al., 1978). The IHP urogenital abnormalities do not conform to normal Mendelian inheritance patterns, and selective breeding of nonafflicted parents does not alter the percentage of afflicted offspring (Cramer and Gill, 1975). Recent work by Shull et al. (2006) has demonstrated that the Renag1 gene on chromosome 14 in the ACI rat is responsible for the incomplete dominance and incomplete penetrance of urogenital inheritance in cross breeding studies. Kneidl et al. (1995) also indicated that afflicted male ACI rats developed genetic abnormalities of chromosome 8 including trisomy and isochromosome.
The ACI rat is very sensitive to exposure to exogenous estrogen or estrogen-mimicking compounds (Shull et al., 1997; Inaguma et al., 2003) and has a marked propensity for the development of spontaneous tumors (Maekawa and Odashima, 1975). Because of this, the ACI rat has made a resurgence into the realm of research in the past decade and has been used in endocrine disruption (Inaguma et al., 2003), breast (Shull et al., 2001; Schaffer et al., 2006; Ravoori et al., 2007), and prostate cancer research (Ward et al., 1980; Varma and Austin, 1990; Kondo et al., 1994; Reyes et al., 2005; Yamashita et al., 2005). ACI rats have been shown to be an applicable model for studying the age-related development of prostate cancer (Reyes et al., 2005). At 24 months of age, 25% of male ACI rats developed intraalveolar atypical hyperplasia (similar in histology to human high grade prostate intraepithelial neoplasia), while at 33 months of age, nearly 100% of the ACI rats developed extensive alveolar prostatic adenocarcinoma with a limited number developing ductal infiltration (Ward et al., 1980).
An extensive search of the literature indicated that there were very limited data on the effect of IHP on the developing prostate. During fetal development, the WD are stabilized by androgens produced in the testes. The prostate is dependent on the diffusion of testicular androgens through the caudal WDS (ED and VD) for development, growth, and stabilization. Development of the prostate is initiated through budding of the urogenital sinus (UGS) urothelium in response to an androgen-dependent secretion of growth factors by the periurethral mesenchyme (Cunha, 1994). Therefore, any perturbation of the WDS could limit the levels of androgens necessary for induction of development and stabilization of the prostate. Using computer-assisted morphometric reconstruction technology (Winsurf®, University of Hawaii), we have examined the prostate of normal and IHP afflicted fetal ACI male rats. To the best of our knowledge, this is the first report of fetal ACI rat prostate reconstruction and illustration of the growth abnormalities in response to altered Wolffian duct development. Our data indicate that a significantly smaller prostate is associated with IHP of the Wollfian ducts in fetal ACI rats. This growth alteration has the potential to alter the postnatal development and growth of the prostate and creates an additional variable that should be considered when using the ACI rat as a primary rodent model for prostate research.
MATERIALS AND METHODS
Time-mated 8- to 10-week old female ACI/SegHSD rats (Harlan Sprague Dawley, Inc. Dublin, VA) arrived at Harlan Biotech Center on gestational day 3 (GD 3). On GD 21, pregnant females were euthanized by CO2 asphyxiation, followed by cervical dislocation. Because it is known that intrauterine position can affect hormonal levels within the UGS, only male fetuses located between a female and male littermate (FMM, 1 M) were selected for this study (Timms et al., 1999; 2005). The 1 M male fetuses were removed via cesarean section, decapitated, and bisected at the level of umbilicus. The caudal portion of the fetuses was placed in individual 50-mL centrifuge tubes containing 10 mL of 4% buffered paraformaldehyde for 2 hr, transferred to fresh paraformaldehyde overnight prior to dehydration in 50% ethanol, and stored at 4°C in 70% ethanol until transferred to the University of South Dakota for final tissue preparation. Upon receipt at the University of South Dakota, the specimens were trimmed of excess material (limbs, tail, and visceral organs) to facilitate embedding and sectioning. Specimens were briefly refixed in 4% buffered paraformeldahyde, dehydrated through graded ethanol and CitriSolv (Fisher Scientific, Pittsburgh, PA), and embedded in paraffin wax in preparation for three-dimensional (3-D) reconstruction.
This study was reviewed and approved by the Harlan Institutional Animal Care and Use Committee (IACUC). Harlan Biotech Center is a fully accredited AAALAC facility. This study complied with all applicable sections of the Guide for the Care and Use of Laboratory Animals (National Research Council, 1997, ISRN C-309-05377-3).
As previously published (Timms et al., 2005; Hofkamp et al., 2008), paraffin-embedded tissues were serially sectioned at 7 μm, floated on a warm water bath, placed on Fisherbrand ColorFrost Plus® slides (Fisher Scientific), and briefly stained with hematoxylin and eosin. Digital photomicrographs were taken using an Olympus DP70 camera attached to an Olympus BX60 microscope (Leeds Precision Instruments, Minneapolis, MN). Histological images of the prenatal prostate and associated secondary sex structures were traced, contoured, and realigned as surfaced-rendered 3-D models using Winsurf® software (University of Hawaii). Morphometric and volumetric analysis of normal and IHP animals were performed and compared to evaluate possible effects on fetal prostate morphology.
All volumetric data were analyzed using a two-tailed t test, assuming equal variances (homoscedastic) comparing normal to IHP animals using Microsoft Excel® software. The confidence interval for rejecting the null hypothesis was P < 0.05. Data are presented as mean ± standard error of the mean (SE). For all groups, n = 4 with all observations and analysis were performed in a blinded fashion to obviate bias.
The developing accessory sex glands (ASGs) consist of anatomically separate structures including the coagulating gland (CG), SV, VD, and the dorsal, lateral, and ventral prostate lobes (DLVPs) (Hayashi et al., 1991; Timms et al., 1994, 2008). Throughout the study, the terms normal and control will be used synonymously to describe fetal male ACI rats without IHP of the Wolffian-derived structures. The combined DLVPs will be referred to as prostate. The SV and VD are formed as derivatives of the Wolffian ducts, whereas the prostate develops from the urothelium of the UGS during early fetal development (Hannema and Hughes, 2007). The SV and VD join together to form the ejaculatory ducts (EJ) (Fig. 1A,D,E,G). Many studies of the adult rodent prostate combine the dorsal and lateral lobes as the dorsolateral prostate. As previously published (Hofkamp et al., 2008), we were able to separate the dorsolateral prostate into its dorsal and lateral lobes and further differentiate the dorsal lobe into distinct cranial and caudal regions (Fig. 1A).
During the course of the experiment, ∼ 25% of the male ACI rats sectioned exhibited the anatomical and histological characteristics of IHP, including the unilateral absence of one SV, VD, ED, and ureter (Fig. 1A–C). The IHP was observed on the right side in all cases. The kidneys were not sectioned or examined and, therefore, no data are available on the prevalence of hydronephrosis.
Accessory Sex Structures
In animals affected with IHP, the volume of the single SV was significantly less (39%; P < 0.05) than the mean volume of the two SVs in the unaffected control animals (Fig. 2A). The mean volume of the CGs in the IHP animals was also significantly less (33%; P < 0.05) than that of normal animals (Fig. 2A). As in previous publications, the volume of the VD was not calculated (Timms et al., 2005; Hofkamp et al., 2008).
The total prostate volume (DLVP; Fig. 1A,F), including the combined DLVPs, in the IHP animals decreased by 40% (P < 0.005; Fig. 2A) when compared with normal male ACI rats. When the dorsal and lateral lobes of the prostate were examined separately, decreases in volume were detected to be 37% (P < 0.001) and 41%, respectively (P < 0.05; Fig. 2B). In the IHP animals, the caudal region of the dorsal lobe was decreased in volume by 32% (P < 0.005), whereas the cranial region of the dorsal lobe was decreased by more than 62% (P < 0.05; Figs. 1A,F and 2B).
Histological analysis of the testes revealed abnormalities ipsilateral to the IHP (Fig. 3D–F). The interior of the ipsilateral testis was primarily composed of connective tissue and was devoid of normal developing seminiferous tubules (Fig. 3C,D). The tunica of the ipsilateral testis appeared normal. The contralateral testis exhibited normal development (Fig. 3F) similar to that of control animals (Fig. 3A–C).
The ACI rat has been a model of IHP, urogenital abnormalities, and renal agenesis for over a half century (Deringer and Heston, 1956). Its usefulness as a model for human renal agenesis has been known for over a quarter of a century (Fisher et al., 1982). As the ACI rat ages, it consistently develops spontaneous tumors of multiple organs, including but not limited to the testes, pituitary, uterus, mammary, and prostate (Maekawa and Odashima, 1975; Ward et al., 1980). Solleveld and Boorman (1986) have previously discussed the problems associated with the use of animals with renal and urogenital abnormalities in long-term toxicological or carcinogenesis studies. Therefore, in this study, we hypothesize that there would be anatomical and developmental difference in the prostate and ASGs of male animals affected by IHP compared with normal male ACI rats. This was confirmed through the use of 3-D reconstruction analysis.
The prostate (DLVP) in the animals with IHP was significantly smaller (40%; P < 0.005) in comparison with normal males. As in prior studies (Hofkamp et al., 2008), the dorsal (37%; P < 0.001) and lateral prostate lobes (41%; P < 0.05) exhibited significant decreases in volume. The caudal region of the dorsal lobe exhibited a significant sensitivity in terms of decreases in volume (32%; P < 0.005) and the number of ducts (26%; P < 0.05), whereas the number of ducts developing from the cranial portion of the dorsal lobe was relatively unaffected (Fig. 4). The single SV contralateral to the aberration in the animals with IHP was significantly smaller, and the Wolffian mesenchyme ipsilateral to the affected side is severely truncated (tWDM; Figs. 1A–D and 2A). Therefore, we conclude that the volumetric changes seen in the animals are a result of a decrease in the size of the solid epithelial prostate buds coupled with a decrease in the number of prostatic buds.
Examination of the ipsilateral and contralateral testes in normal and IHP animals revealed striking histological differences that may contribute to the potential mechanism of the urogenital abnormalities described in this study (Fig. 3). In the unaffected animals, the ED is present, and the testes have histologically normal architecture and seminiferous tubule development (Fig. 3A–C). In the ipsilateral testis of the IHP-affected animal, the seminiferous tubules are absent. The body of the testis beneath the tunica albuginea in the IHP animals was composed of loose connective tissue (Fig. 3D,E). The contralateral testis of the IHP-affected animals exhibit normal testicular histology (Fig. 3D,F). In the affected animals, ipsilateral to the urogenital abnormalities there is a complete loss of the ED (Fig. 3D,E), whereas contralateral to the abnormalities the ED is present and histologically normal (Fig. 3D,F). Prior studies have shown that unilateral castration causes unilateral WD regression (Hannema and Hughes, 2007). The observation of altered development of the testis and ED ipsilateral to the abnormalities in this study may be associated with asymmetric development of the UGS in the animals with IHP. In contrast with our finding of fetal testicular abnormalities, previous publications on ACI rat IHP only reported changes after puberty (Marshall et al., 1979, 1982; McCullough et al., 1984). Therefore, while ipsilateral changes in the testis and WD-derived structures provide a potential mechanism of causality, further research is needed to completely understand and confirm the mechanisms responsible for the prostate development and growth abnormalities seen in the animals with IHP.
The origins of the prostatic utricle have been debated for a century (Meyer, 1909; Shapiro et al., 2004). The prostatic utricle was originally thought to be a nonregressed Müllerian remnant. However, Shapiro et al. (2004) concluded that the utricle was of definite UGS origin. In our current study, the utricle of animals with IHP exhibited both a histologically (Fig. 1E,G) and developmentally abnormal morphology (Fig. 1A,B,D,F,H). Following reconstruction and volumetric analysis, the absolute volume of the utricle within the fetal ACI rat with IHP was found not to differ significantly from that of normal males (data not shown).
Although there was no significant change in the absolute volume of the utricle in IHP ACI rats, there was an interesting anatomical difference in utricle histology and morphology. In the normal animal, the utricle joins to the urothelium in two places in the dorsal region of the seminal colliculus (Timms et al., 1994). This connection creates a tissue bridge that is characteristic of the utricle in the developing prostate (Fig. 1G, H; Shapiro et al., 2004). This area is associated with a dense mesenchyme reminiscent of vaginal mesenchyme (Kurita and Cunha, 2001; Shapiro et al., 2004). However, in the IHP animals, there was only a single side entrance contralateral to the affected side (Fig. 1G,H). The IHP utricle was also curved toward to the side lacking SV and VD, instead of forming a linear structure flanked by the EJs (Fig. 1D,E,H). The curvature of the utricle in IHP animals gives the initial impression of a truncated EJ or uterine horn (Fig. 1E). In the female, this mesenchyme is essential for the formation of the Müllerian-derived vaginal epithelium. Within the male ACI rat, there is a dense area of vaginal-like mesenchyme (VLM) dorsal to the utricle bridge in the seminal colliculus in the normal ACI rat (Fig. 1G, top panel). In the IHP animals, the histological extent of the VLM is more diffuse as it surrounds the single entrance of the utricle into the urothelial wall (Fig. 1G, lower panel).
This study was prompted by the recommendation proposed by the Prostate Cancer Foundation at the Prostate Cancer Models Working Group and their request that all currently available animal models of prostate cancer be characterized with respect to their usefulness to researchers and their analogy to human cancer (Pienta et al., 2008). The ACI rat is a fundamental research model for the study of renal agenesis and age-related cancer of multiple tissues. Its inherent sensitivity to exogenous estrogen exposure and extensive development of breast and prostate adenocarcinoma has made it an invaluable tool for researchers. However, because of the abnormalities seen in the fetal ACI rat prostate, and in agreement with previous publications (Solleveld and Boorman, 1986), we recommend that care should be taken when selecting the ACI rat as the predominant model for endocrine disruption or age-related cancer research on the prostate. Alterations to the developing fetal prostate can have implications far into adulthood (Prins et al., 2008). Further studies are needed to elucidate the molecular mechanisms responsible for the ipsilateral abnormalities.
The authors thank Ellen Shapiro, M.D., for her critical review of the manuscript, Eric Neufeld, Ph.D., for providing specific upgrades to the Winsurf® software, Krista Hofkamp for her help in completing the reconstructions, J. Kevin Kerzee, Ph.D., for his work as study director, and Lisa Taitt for her work as the study coordinator at the Harlan Biotech Center.