A. Donjacour

Hormonal, cellular, and molecular control of prostatic development

The prostate is a male accessory sex gland found only in mammals that functions to produce a major fraction of seminal fluid. Interest in understanding the biology of the prostate is driven both by the fascinating nature of the developmental processes that give rise to the prostate and by the high incidence in humans of prostatic diseases, including prostatic adenocarcinoma and benign prostatic hyperplasia. This review summarizes the current state of knowledge of the cellular and molecular processes that control prostatic development. Insight into the mechanisms that control prostatic development has come from experimental embryological work as well as from the study of mice and humans harboring mutations that alter prostatic development. These studies have demonstrated a requirement for androgens throughout prostatic development and have revealed a series of reciprocal paracrine signals between the developing prostatic epithelium and prostatic mesenchyme. Finally, these studies have identified several specific gene products that are required for prostatic development. While research in recent years has greatly enhanced our understanding of the molecular control of prostatic development, known genes cannot yet explain in molecular terms the complex biological interactions that descriptive and experimental embryological studies have elucidated in the control of prostatic development.

Hormonal, cellular, and molecular regulation of normal and neoplastic prostatic development.

This review on normal and neoplastic growth of the prostate emphasizes the importance of epithelial-mesenchymal/stromal interactions. Accordingly, during prostatic development urogenital sinus mesenchyme (a) specifies prostatic epithelial identity, (b) induces epithelial bud formation, (c) elicits prostatic bud growth and regulates ductal branching, (d) promotes differentiation of a secretory epithelium, and (e) specifies the types of secretory proteins expressed. In reciprocal fashion, prostatic epithelium induces smooth muscle differentiation in the mesenchyme. Epithelial-mesenchymal interactions during development continue postnatally into adulthood as stromal-epithelial interactions which play a homeostatic role and in so doing reciprocally maintain epithelial and stromal differentiation and growth-quiescence. Prostatic carcinogenesis involves perturbation of these reciprocal homeostatic cell-cell interactions. The central role of mesenchyme in prostatic epithelial development has been firmly established through analysis of tissue recombinants composed of androgen-receptor-positive wild-type mesenchyme and androgen-receptor-negative epithelium. These studies revealed that at the very least ductal morphogenesis, epithelial cytodifferentiation, epithelial apoptosis and epithelial proliferation are regulated by stromal and not epithelial androgen receptors. Likewise, progression from non-tumorigenesis to tumorigenesis elicited by testosterone plus estradiol proceeds via paracrine mechanisms. Thus, stromal-epithelial interactions play critical roles in the hormonal, cellular, and molecular regulation of normal and neoplastic prostatic development.

Paracrine regulation of apoptosis by steroid hormones in the male and female reproductive system

In males, androgens are essential in maintaining the integrity of the prostate. Androgen-ablation induces apoptosis of the prostatic epithelium. In females, ovariectomy induces apoptosis in uterine epithelium while progesterone inhibits this process. The objective of this study was to determine whether androgen and progesterone inhibit apoptosis, respectively, in mouse prostatic and uterine epithelia via steroid receptors in the epithelium or in the stroma. To address this question, prostatic tissue recombinants were prepared with rat urogenital sinus mesenchyme plus bladder epithelium from wild-type or testicular feminization mutant (Tfm) mice. Thus, prostatic tissue was generated having androgen receptor (AR) in both epithelium and stroma or in the stroma only. Castration of hosts induced apoptosis in the AR-negative Tfm prostatic epithelium with an epithelial apoptotic index virtually identical to prostatic tissue recombinants containing wild-type epithelium. Moreover, this castration-induced prostatic epithelial apoptosis was blocked by testosterone and dihydrotestosterone in both wild-type and Tfm prostatic tissue recombinants. Likewise, uterine tissue recombinants were prepared in which epithelium and/or stroma was devoid of progesterone receptor (PR) by using uterine epithelium and stroma of wild-type and PR knockout mice. Progesterone inhibited uterine epithelial apoptosis only in tissue recombinants prepared with PR-positive stroma. The PR status of the epithelium did not affect epithelial apoptotic index. Therefore, the apoptosis in prostatic and uterine epithelia is regulated by androgen and progesterone via stromal AR and PR, respectively. In both cases, epithelial AR or PR is not required for hormonal regulation of epithelial apoptosis in prostatic and uterine epithelium.

Rapid Inhibition of Cancer Cell Growth Induced by Lentiviral Delivery and Expression of Mutant-Template Telomerase RNA and Anti-telomerase Short-Interfering RNA

In human cancers, telomeres are commonly maintained by elevated levels of the ribonucleoprotein enzyme telomerase, which contains an intrinsic templating RNA moiety (human telomerase RNA; hTER) and the core protein (human telomerase reverse transcriptase). We developed a lentiviral system for efficient overexpression of mutant-template human telomerase RNA (MT-hTer) to add mutant DNA to telomeres in cancer cells. We show that such MT-hTer overexpression rapidly inhibits cell growth and induces apoptosis in telomerase-positive precancerous or cancer cells but not in telomerase-negative cells. These rapid effects occurred independent of wild-type p53 and telomere length. Tumor growth and progression were significantly decreased in xenografts of human tumor cells overexpressing MT-hTers. Expression of a hairpin short-interfering RNA that specifically targeted the endogenous wild-type hTER template region, but spared the MT-hTers, also caused p53-independent cell growth inhibition and apoptosis, and when coexpressed with MT-hTer, synergistically killed cancer cells. Hence, anti-wild-type-hTER short-interfering RNA and MT-hTers may act through distinct pathways and, particularly in combination, represent a promising approach to anticancer therapies.

Stromal regulation of epithelial function

Stromal influences upon epithelia are part of a continuum of cellular interactions that begins at fertilization and extends into adulthood. In parenchymal organs, the most thoroughly characterized interactions have been those that occur during development between mesenchyme, embryonic stroma, and epithelium. Mesenchyme is essential for epithelial proliferation, morphogenesis, and differentiation. Hormones affect stromal-epithelial interactions, and in some cases, steroid hormones may produce their effects on the epithelium indirectly, acting via the mesenchyme. In many adult organs the epithelia continually proliferate and differentiate and consequently may be considered developing systems within the mature organism. This is especially true in organs with a rapidly renewing epithelium, such as the intestine, and in organs that have cycles of functional activity, such as those of the female reproductive system. The mechanisms by which stroma affects epithelial structure and function are not well understood. Current models of how signaling may be accomplished include transmission via diffusible substances, via the extracellular matrix (ECM), and via direct cell-cell contact. Growth factors and organ-specific paracrine factors are candidates for stromal cues that affect the epithelium in some systems. Components of the ECM appear to play a role in permissive interactions and may affect epithelial function by changing cell shape or by binding ECM to the cell surface integrin receptors. Signaling via direct stromal-epithelial contact may be accomplished via interactions between complimentary cell surface adhesion molecules. The importance of stromal-epithelial interactions is reemphasized by several models of carcinogenesis that suggest that perturbations in these interactions may be involved in tumor progression.

FGF-10 plays an essential role in the growth of the fetal prostate

Induction and branching morphogenesis of the prostate are dependent on androgens, which act via the mesenchyme to induce prostatic epithelial development. One mechanism by which the mesenchyme may regulate the epithelium is through secreted growth factors such as FGF-10. We have examined the male reproductive tract of FGF-10(-/-) mice, and at birth, most of the male secondary sex organs were absent or atrophic, including the prostate, seminal vesicle, bulbourethral gland, and caudal ductus deferens. Rudimentary prostatic buds were occasionally observed in the prostatic anlagen, the urogenital sinus (UGS) of FGF-10(-/-) mice. FGF-10(-/-) testes produced sufficient androgens to induce prostatic development in control UGS organ cultures. Prostatic rudiments from FGF-10(-/-) mice transplanted into intact male hosts grew very little, but showed some signs of prostatic differentiation. In cultures of UGS, the FGF-10 null phenotype was partially reversed by the addition of FGF-10 and testosterone, resulting in the formation of prostatic buds. FGF-10 alone did not stimulate prostatic bud formation in control or FGF-10(-/-) UGS. Thus, FGF-10 appears to act as a growth factor which is required for development of the prostate and several other accessory sex organs.

Effect of in vitro fertilization on gene expression and development of mouse preimplantation embryos

In vitro culture (IVC) of preimplantation mouse embryos is associated with changes in gene expression. It is however, not known if the method of fertilization affects the global pattern of gene expression. We compared gene expression and development of mouse blastocysts produced by in vitro fertilization (IVF) versus blastocysts fertilized in vivo and cultured in vitro from the zygote stage (IVC) versus control blastocysts flushed out of the uterus on post coital day 3.5. The global pattern of gene expression was assessed using the Affymetrix 430 2.0 chip. It appears that each method of fertilization has a unique pattern of gene expression and development. Embryos cultured in vitro had a reduction in the number of trophoblastic cells (IVF 33.5 cells, IVC 39.9 cells, and 49.6 cells in the in vivo group) and, to a lesser degree, of inner cell mass cells (12.8, 11.7, and 13.8 respectively). The inner cell mass nuclei were larger after culture in vitro (140 microm(2), 113 microm(2), and 86 microm(2) respectively). Although a high number of genes (1912) was statistically different in the IVF cohort when compared with the in vivo control embryos, the magnitude of the changes in gene expression were low and only a minority of genes (29 genes) was changed more than fourfold. Surprisingly, IVF embryos were different from IVC embryos (3058 genes were statistically different, but only three changed more than fourfold). Proliferation, apoptosis, and morphogenetic pathways are the most common pathways altered after IVC. Overall, IVF and embryo culture have a profound effect on gene expression pattern and phenotype of mouse preimplantation embryos.

The effect of androgen deprivation on branching morphogenesis in the mouse prostate

Androgen-induced prostatic development encompasses many individual processes such as ductal branching morphogenesis, cellular proliferation, and secretory cytodifferentiation. Previous studies of ductal morphogenesis (Y. Sugimura, G.R. Cunha, and A.A. Donjacour, 1986, Biol. Reprod. 34, 961-971) demonstrated that the majority (approximately 70%) of ductal tips and branchpoints in the mouse prostate is generated before 15 days of age. Since circulating androgen levels are low during this neonatal period, it is possible that ductal branching morphogenesis may not require the continuous presence of androgens. To test this hypothesis mice were castrated within 24 hr of birth, and prostates from these mice were microdissected at various ages from 5 to 120 days of age to assess the number of ductal tips and branchpoints; wet weight and DNA content were also determined. In intact males wet weight and DNA content increased rapidly between 15 and 60 days of age, after most of the prostatic ductal architecture had been laid down. Neonatal castration considerably reduced the number of tips and branchpoints in both the ventral and dorsolateral prostate, yet both lobes still underwent significant branching morphogenesis in the absence of testes. The administration of anti-androgens to neonatal castrates did not suppress ductal branching to any greater extent than did neonatal castration alone. Androgen replacement immediately following neonatal castration resulted in precocious attainment of the adult number of tips and branchpoints, but caused only modest increases in wet weight. In contrast, when androgen replacement was delayed until adulthood, prostatic wet weight increased to normal adult levels, but the number of ductal tips and branchpoints did not. These experiments show that neonatal prostatic ductal morphogenesis is sensitive to, but does not require, chronic androgen stimulation.

Effect of the method of conception and embryo transfer procedure on mid-gestation placenta and fetal development in an IVF mouse model

BACKGROUND Abnormal placentation is a potential mechanism to explain the increased incidence of low birthweight observed after IVF. This study evaluates, in a mouse model, whether the method of conception and embryo transfer affect placentation and fetal development. METHODS IVF blastocysts (CF1 x B6D2F1/J) were cultured in Whittens medium (IVF(WM), n = 55) or K modified simplex optimized medium with amino acids (IVF(KAA), n = 56). Embryos were transferred to the uteri of pseudo-pregnant recipients. Two control groups were created: unmanipulated embryos produced by natural mating (in vivo group, n = 64) and embryos produced by natural mating that were flushed from uterus and immediately transferred to pseudo-pregnant recipients (flushed blastocysts, FB group, n = 57). At gestation age 12.5 days, implantation sites were collected and fixed; fetuses and placentas were weighed and their developmental stage (DS) evaluated. Placental areas and vascular volume fractions were calculated; parametric statistics were applied as appropriate. RESULTS IVF fetuses showed a modest but significant delay in development compared with FB mice (P < 0.05). In addition, IVF conceptuses were consistently smaller than FB (P < 0.05). Importantly, these differences persisted when analyzing fetuses of similar DS. The placenta/fetus ratio was larger in the IVF group (IVF(WM) 0.95; IVF(KAA) = 0.90) than the FB group (0.72) (P < 0.05 for all comparisons). Gross morphology of the placenta and ratio labyrinth/fetal area were equivalent in the IVF and FB groups, as were percentage of fetal blood vessels, maternal blood spaces and trophoblastic components. CONCLUSIONS In vitro embryo culture affects fetal and placental development; this could explain the lower birthweight in IVF offspring.

Growth, regeneration, and tumorigenesis of the prostate activates the PSCA promoter

The prostate gland undergoes dramatic changes in growth status during normal physiologic development, following androgen administration to castrate animals, and during tumor development. The prostate stem cell antigen (PSCA, named for its strong sequence homology to the thymocyte marker stem cell antigen 2) is a cell surface molecule associated with human and murine prostate cancer. To help define the regulation of this molecule, we created a transgenic mouse strain, which uses the human PSCA promoter region to control the expression of enhanced green fluorescent protein (GFP). Expression of GFP was detected in mid-gestation following the appearance of prostatic buds from the urogenital sinus. In adult mice, GFP expression was restricted to a subset of cells located in the distal tips of the glands. GFP expression increased during puberty and regeneration driven by androgen and associated with expansive growth of the prostate. GFP-positive cells coexpressed markers associated with both basal and secretory cells in the human prostate. Prostate carcinogenesis driven by T antigen in the transgenic adenocarcinoma of the mouse prostate (TRAMP) model results in an increased percentage and intensity level for PSCA promoter-driven GFP-positive cells. This transgenic system helps define the range of cellular changes associated with altered expression of PSCA, shows that transcriptional control is a major component regulating PSCA levels, and provides a useful tool to study subpopulations of prostate epithelial cells and factors that regulate the PSCA promoter.