Barry G. Timms

Ductal Budding and Branching Patterns in the Developing Prostate

Development of the prostate was studied by serial section reconstruction and computer-assisted three-dimensional analysis. A comparison of ductal budding in species of rat and mouse and the human revealed patterns consistent with common developmental characteristics. Ventral, lateral and dorsal lines of epithelial buds, which emanated from the urogenital sinus into the surrounding periurethral mesenchyme, followed ventro-dorsal and cranio-caudal axes. Subsequent branching morphogenesis was associated with specific mesenchymal condensations. These patterns of budding were closely related to the adult lobe architecture in the rodent prostate. In the human fetus, prostate ductal budding exhibited patterns compatible with the current concept of zonal anatomy.

Developmental toxicity of UV filters and environmental exposure: a review

Several ultraviolet (UV) filters exhibit estrogenic, some also anti-androgenic activity. They are present in waste water treatment plants, surface waters and biosphere including human milk, suggesting potential exposure during development. Developmental toxicity was studied in rats for the UV filters 4-methylbenzylidene camphor (4-MBC, 0.7, 7, 24, 47 mg/kg/day) and 3-benzylidene camphor (3-BC, 0.07, 0.24, 0.7, 2.4, 7 mg/kg/day) administered in chow to the parent generation before mating, during pregnancy and lactation, and to the offspring until adulthood. Neonates exhibited enhanced prostate growth after 4-MBC and altered uterine gene expression after both chemicals. 4-MBC and 3-BC delayed male puberty and affected reproductive organ weights of adult offspring. Effects on the thyroid axis were also noted. Expression and oestrogen sensitivity of oestrogen-regulated genes and nuclear receptor coregulator levels were altered at mRNA and protein levels in adult uterus, prostate and brain regions involved in gonadal control and sexual behaviour. Female sexual behaviour was impaired by both filters; 3-benzylidene camphor caused irregular cycles. Classical endpoints exhibited lowest observed adverse effect levels (LOAELs) and no observed adverse effect levels (NOAELs) of 7/0.7 mg/kg for 4-MBC and 0.24/0.07 mg/kg for 3-BC. Molecular endpoints were affected by the lowest doses studied. Our data indicate that the potential risk posed by endocrine active UV filters warrants further investigations.

PROSTATE GLAND GROWTH DURING DEVELOPMENT IS STIMULATED IN BOTH MALE AND FEMALE RAT FETUSES BY INTRAUTERINE PROXIMITY TO FEMALE FETUSES

In rodents, steroid hormones are transported between adjacent fetuses, and male or female fetuses that develop in utero between female fetuses (2F males or 2F females) have higher serum levels of estradiol and lower serum levels of testosterone relative to siblings of the same sex that develop between two male fetuses (2M males or 2M females). The present study was prompted by the prior unexpected finding that as adults, 2F male mice have an enlarged prostate, and increased numbers of prostatic androgen receptors relative to 2M males. We examined prostate development in both male and female rat fetuses from different intrauterine positions using computer-assisted, 3-dimensional reconstruction of the urogenital complex. In males, this included the prostate, seminal vesicles and utricle (a remnant of the Müllerian ducts), while in females it included development of prostatic glandular buds. The mean cross-sectional area of developing prostatic epithelial buds, utricle and seminal vesicles was significantly increased in 2F male relative to 2M male fetuses. In female fetuses, prostatic bud development was significantly more likely to occur in 2F (67%) than in 2M (29%) animals. These findings suggest that the transport of a small supplement of estrogen from adjacent female fetuses enhances androgen-dependent accessory organ development. We also found that mRNAs encoding receptors for both estrogen and androgen were located in the mesenchyme of the developing male prostate. The localization of estrogen and androgen receptor mRNA in this region further suggests that the mesenchymal induction of prostatic epithelial growth involves both hormones. The cranial dorsolateral prostatic buds exhibited the greatest enlargement in 2F males. This region of the developing prostate in rats is comparable (that is the embryonic homologue) to the region exhibiting benign prostatic hyperplasia (BPH) during aging in men. We propose that the potential for pathological regrowth of the prostate during aging is imprinted by estradiol during fetal development.

Prostate development: a historical perspective

The regional anatomy of the human prostate has been debated periodically over the last century with various levels of controversy and agreement, beginning with the concept of lobes and replaced by the current model of zones. During this period a variety of classifications have been proposed, based upon the studies of glandular morphogenesis, responses to hormones or histopathology. The current paradigm suggests that the regional differences seen in the prostate of both animal models and the human are a consequence of specific epithelial-mesenchymal interactions along the cranial-caudal axis of the urogenital sinus. The distinctive regional patterns seen in the rodent prostate and the histological heterogeneity of the human adult gland all point to the modification of the distal portion of the ducts, while the proximal segments retain their spatial relationship to the urethra that was formed during fetal development. This suggests that the early epithelial budding that occurs in utero represents a common, fairly symmetrical pattern of growth in many species, while the regional differences in branching morphogenesis and cytodifferentiation are controlled by the instructional influences of mesenchyme and temporal expression of growth factors. Perturbation of the normal processes involved during critical periods of fetal development during reproductive organ development may also play a role in the susceptibility of the prostate to disease in adulthood. Past descriptions of detailed anatomical studies, which span over a century, have provided much insight into the architecture and processes that form a complex tubulo-alveolar gland. New insights into the ductal detail and the advent of sophisticated analyses of cell-cell interactions and molecular mechanisms controlling pathways of cellular growth, differentiation, and apoptosis will likely lead to new approaches for prevention and therapy of prostatic diseases.

Prostate development and growth in benign prostatic hyperplasia

The etiology of benign prostatic hyperplasia [BPH] in elderly men has intrigued anatomists, pathologists and scientists for centuries. Studies of morbid anatomy, clinical observations and contemporary cellular biology have contributed to an evolving interpretation of the causality of the disease. Insights into the detailed microanatomy and ductal architecture of the prostate during stages of fetal and early postnatal development suggest that mechanisms involved in the early growth period become aberrantly expressed in elderly men. Age, hormones and epithelial-mesenchymal interactions are all contributing factors to the pathogenesis of BPH. Control of the microenvironment in normal and abnormal growth is a multifactorial process. Susceptibility to the disease may include clinical comorbid diseases, region-specific changes in cell-cell interactions and a variety of signaling pathways including a novel hypothesis regarding the role of the primary cilium as a regulator of signal transduction mechanisms. Recent work in animal models has shown that there are region-specific differences within the prostate that may be significant because of the dynamic and intricate interplay between the epithelium and mesenchyme. Because of the focal nature of BPH a closer examination of normal morphogenesis patterns, which defines the glands architecture, may facilitate a detailed understanding of the atypical growth patterns.

Testosterone and 17β-Estradiol Induce Glandular Prostatic Growth, Bladder Outlet Obstruction, and Voiding Dysfunction in Male Mice

Benign prostatic hyperplasia (BPH) and bladder outlet obstruction (BOO) are common in older men and can contribute to lower urinary tract symptoms that significantly impact quality of life. Few existing models of BOO and BPH use physiological levels of hormones associated with disease progression in humans in a genetically manipulable organism. We present a model of BPH and BOO induced in mice with testosterone (T) and 17β-estradiol (E(2)). Male mice were surgically implanted with slow-releasing sc pellets containing 25 mg T and 2.5 mg E(2) (T+E(2)). After 2 and 4 months of hormone treatment, we evaluated voiding patterns and examined the gross morphology and histology of the bladder, urethra, and prostate. Mice treated with T+E(2) developed significantly larger bladders than untreated mice, consistent with BOO. Some mice treated with T+E(2) had complications in the form of bladder hypertrophy, diverticula, calculi, and eventual decompensation with hydronephrosis. Hormone treatment caused a significant decrease in the size of the urethral lumen, increased prostate mass, and increased number of prostatic ducts associated with the prostatic urethra, compared with untreated mice. Voiding dysfunction was observed in mice treated with T+E(2), who exhibited droplet voiding pattern with significantly decreased void mass, shorter void duration, and fewer sustained voids. The constellation of lower urinary tract abnormalities, including BOO, enlarged prostates, and voiding dysfunction seen in male mice treated with T+E(2) is consistent with BPH in men. This model is suitable for better understanding molecular mechanisms and for developing novel strategies to address BPH and BOO.

Region-specific growth effects in the developing rat prostate following fetal exposure to estrogenic ultraviolet filters.

Background and objectives Exposure to environmental endocrine disruptors is a potential risk factor for humans. Many of these chemicals have been shown to exhibit disruption of normal cellular and developmental processes in animal models. Ultraviolet (UV) filters used as sunscreens in cosmetics have previously been shown to exhibit estrogenic activity in in vitro and in vivo assays. We examined the effects of two UV filters, 4-methylbenzylidene camphor (4-MBC) and 3-benzylidene camphor (3-BC), in the developing prostate of the fetal rat. Methods Pregnant Long Evans rats were fed diets containing doses of 4-MBC and 3-BC that resulted in average daily intakes of these chemicals corresponding to the lowest observed adverse effects level (LOAEL) and the no observed adverse effects level (NOAEL) doses in prior developmental toxicity studies. Using digital photographs of serial sections from postnatal day 1 animals, we identified, contoured, and aligned the epithelial ducts from specific regions of the developing prostate, plus the accessory sex glands and calculated the total volume for each region from three-dimensional, surface-rendered models. Results Fetal exposure to 4-MBC (7.0 mg/kg body weight/day) resulted in a significant increase (p < 0.05) in tissue volume in the prostate and accessory sex glands. Treated males exhibited a 62% increase in the number of ducts in the caudal dorsal prostate. Increased distal branching morphogenesis appears to be a consequence of exposure in the ventral region, resulting in a 106% increase in ductal volume. Conclusions 4-MBC exposure during development of the male reproductive accessory sex glands exhibited classical growth effects associated with estrogenic endocrine disruptors. The different regional responses suggest that the two developmental processes of ductal outgrowth and branching morphogenesis are affected independently by exposure to the environmental chemicals.

Isolation and characterization of rabbit ovarian surface epithelium, granulosa cells, and peritoneal mesothelium in primary culture.

SummaryMammalian ovarian surface epithelial (OSE) cells and peritoneal mesothelial (PM) cells have a common embryologic origin, yet certain morphologic and histochemical characteristics are different in the adult. In this study, a two-step culture method was developed to examine the characteristics of these two cell types in vitro. OSE, PM, and ovarian granulosa (GC) cells were isolated from estrous rabbits and cultured for 6 d in 5% serum-supplementedd-valine medium (to inhibit fibroblast growth), then incubated for a further 2 d in serum-free McCoys 5A medium. This study showed that rabbit OSE and PM cells in vitro maintained certain in vivo morphologic characteristics; OSE cells exhibited distinct cell borders and abundant microvilli of homogeneous size and shape, whereas PM cells were characterized by obscure cell borders and abundant microvilli of heterogeneous form. GC in vitro exhibited overlapping cell borders and sparse microvilli of homogeneous structure. This study showed for the first time that cultured rabbit OSE and PM cells, but not GC, contain distinct filaments of cytokeratin 18. In addition, rabbit OSE cells and GC, but not PM cells, contained 17β-hydroxysteroid dehydrogenase. However, only GC contained delta 5-3β hydroxysteroid dehydrogenase. OSE, PM, and GC maintained their ultrastructural and histochemical characteristics in serum-free medium. These results suggest that rabbit OSE cells in vitro could be distinguished from PM cells by histochemical and ultrastructural differences. Furthermore, because these characteristics were not altered in serum-free medium, the two-step culture method will be valuable in further hormonal studies of these cells in vitro.

Novel expression patterns of the myc/max/mad transcription factor network in developing murine prostate gland.

PURPOSE Expression of myc proto-oncogenes and myc-antagonizing mad/mxi genes typically predominate in proliferating versus differentiating cells, respectively. C-myc expression in prostate cells is well established but to our knowledge that of several recently discovered mad/mxi genes is completely uncharacterized. Such characterization is particularly relevant because mxi1 is lost or mutated in some human prostate tumors and mouse mxi1-null mutants show prostatic hyperplasia. MATERIALS AND METHODS Developing murine prostatic lobes at select postnatal days 1 to 28 were analyzed by in situ immunohistochemical and in vitro RNA analysis. The expression patterns of the 3 myc genes c-, L- and N-myc, and the mad1, mxi1 and mad4 genes were studied in most detail with nonradioactive in situ and immunohistochemical analyses. RESULTS We describe what is to our knowledge previously unreported expression of N- and L-myc in the prostate with particularly the latter strongly expressed throughout development. High c-myc expression was lost at day 7 with re-elevation at day 14, followed by subsequent low expression, representing a unique in vivo confirmation of c-myc expression changes seen previously in several in vitro differentiation systems. The alternatively spliced weak and strong repressor mxi1 isoforms showed distinct, partially overlapping expression patterns. Of particular interest were continual mad1 and mad4 expression during the proliferative and differentiative phases. Similarly mad1 was evident in proliferating normal prostate cell cultures but not in tumor cell lines, suggesting that mad1 expression in prostate may be clinically relevant. CONCLUSIONS Myc network expression in developing mouse prostate is novel and does not completely fit previous simpler models of Myc versus Mad expression based on other cell types.

Atypical fetal prostate development is associated with ipsilateral hypoplasia of the wolffian ducts in the ACI rat.

For over a half century, the ACI (August × Copenhagen) rat has been a primary model for studying renal agenesis and ipsilateral hypoplasia (IHP) of the Wolffian‐derived structures (WDS). Because the ACI rat is also used as a model for prostate research, it is important to examine the relationship of IHP and urogenital sinus (UGS) development. The prostate is dependent on androgens for proper growth and differentiation. Alteration in androgen production and/or delivery to the UGS has the potential to perturbate normal development. In this study, we investigate whether the ipsilateral loss of the WDS is associated with altered prostate development. Digital images of serial‐sectioned fetal ACI rat UGS were used to create three‐dimensional (3‐D) surface‐rendered models of the developing prostate, seminal vesicle, vas deferens, and utricle on gestational day 21. The number and volume of prostate ducts developing from the UGS were calculated from the 3‐D model data. Animals exhibiting IHP had a significant decrease in total fetal prostate volume (40%; P < 0.005) with significant regional specific differences when compared with normal male ACI rats. Anatomical and histological differences in the utricle, abnormal histology of the ipsilateral testes, and a truncation of the ipsilateral Wolffian ductal mesenchyme were also seen in the animals with IHP. Additional research is needed to further understand the mechanisms and consequences of IHP on prostate growth and development. Alterations to normal prenatal development of the male accessory sex organs can have important consequences for the growth and morphology of the adult gland. Anat Rec, 2010.