Induction of functional cytodifferentiation in the epithelium of tissue recombinants. I. Homotypic seminal vesicle recombinants

Functional cytodifferentiation of seminal vesicle epithelium was investigated in tissue recombinants. Neonatal rat and mouse seminal vesicles were separated into epithelium and mesenchyme using trypsin. Epithelium and mesenchyme were then recombined in vitro to form interspecific rat/mouse homotypic recombinants. Growth as renal grafts in adult male athymic mice resulted in seminal vesicle morphogenesis in 70% of the recombinants (the remaining 30% failed to grow). Functional cytodifferentiation was judged by the expression of the major androgen-dependent secretory proteins characteristic of the seminal vesicles of adult rats and mice. Antibodies specific for each of these proteins were used to screen tissue sections by immunocytochemistry and to probe protein extracts by immunoblotting techniques. The heterospecific recombinants synthesized the full range of seminal vesicle secretory proteins that typifies the species providing the epithelium of the recombinant, not the mesenchyme. There was little functional variation between individual recombinants. The time course of development corresponded to that of intact neonatal seminal vesicles grown under the same conditions. Morphogenesis and functional cytodifferentiation were not evident after one week, but were well advanced after two weeks. Seminal vesicle recombinants grown for three weeks were indistinguishable morphologically and functionally from normal adult seminal vesicles. In addition, the ability of adult seminal vesicle epithelium to be induced to proliferate was examined. In association with neonatal seminal vesicle mesenchyme, the epithelium of the adult seminal vesicle proliferated and retained its normal functional activity. Thus, seminal vesicle functional cytodifferentiation can be faithfully reproduced in homotypic tissue recombinants. The methods used in this study will be used to investigate seminal vesicle development in instructive inductions of heterotypic epithelia.     

Effects of mesenchyme of the embryonic urogenital sinus and neonatal seminal vesicle on the cytodifferentiation of the Dunning tumor: ultrastructural study.

The aim of the present study was to examine the effects of mesenchyme on the cytodifferentiation of the Dunning tumor (DT, R3327), a transplantable rat prostatic adenocarcinoma developed spontaneously from the dorsolateral prostate of a Copenhagen rat. Small pieces of DT were combined with mesenchyme of the rat urogenital sinus (18-day fetal, UGM) or seminal vesicle (0-day neonatal, SVM). Both types of combinations were grown under the kidney capsule of male athymic nude mice for 4 weeks. At harvest, the tissue recombinants were fixed and processed for electron microscopy. Grafts of parental DT were similarly processed for electron microscopy. The tumor was characterized by tubules lined by 2-3 layers of undifferentiated cells lacking secretory granules. The basal lamina was reduplicated, and epithelioid cells traversing gaps in the basal lamina were frequently observed. The stroma was composed of a mixture of fibroblastic and large epithelioid cells derived from the ductal lining epithelium through a process of micrometastasis. In UGM or SVM+DT combinations the mesenchyme influenced the differentiation and secretory activity of the DT epithelium. The induced DT epithelial cells exhibited a well-developed granular endoplasmic reticulum, a large Golgi apparatus and prominent secretory granules which were never observed in the parental DT. The basal lamina returned to normal, while the incidence of micrometastasis was decreased. The collagen content of the stroma was increased with a concurrent appearance of smooth muscle cells surrounding those tubules where secretory cytodifferentiation had occurred. While the mechanism involved in the mesenchyme-induced change in cytodifferentiation remains unknown, it is evident that the DT epithelial cells when associated with normal embryonic or neonatal mesenchyme can express a more normal cytodifferentiation and function. It is concluded (a) that the DT cells can be induced by mesenchyme to express more highly differentiated ultrastructural patterns and secretory cytodifferentiation, (b) that the induced secretory cytodifferentiation is associated with a reduction in invasiveness (micrometastasis) and a more normal-appearing basal lamina and (c) that the increased abundance of collagen fibers and the differentiation of smooth muscle in the stromal compartment are associated with secretory cytodifferentiation suggesting that reciprocal epithelial-mesenchymal interactions are involved in the regulation of the pathobiology of the DT.     

Neonatal seminal vesicle mesenchyme induces a new morphological and functional phenotype in the epithelia of adult ureter and ductus deferens

Mesenchyme from neonatal mouse and rat seminal vesicles (SVM) was grown in association with postnatal (adult) epithelial cells from the ureter (URE) and ductus deferens (DDE) in chimeric tissue recombinants composed of mouse mesenchyme and rat epithelium or vice versa. Functional cytodifferentiation was examined in these SVM + URE and SVM + DDE tissue recombinants with antibodies against major androgen-dependent seminal-vesicle-specific secretory proteins. Adult DDE and URE were induced to express seminal cytodifferentiation and produced the complete spectrum of major seminal vesicle secretory (SVS) proteins. The SVS proteins produced were specific for the species that provided the epithelium. In the case of SVM + URE recombinants, the URE, which normally lacks androgen receptors (AR), expressed AR. These results demonstrate that adult epithelial cells retain a developmental plasticity equivalent to their undifferentiated fetal counterparts and are capable of being reprogrammed to express a completely new morphological, biochemical and functional phenotype.     

Fine structure of the Wolffian duct and cytodifferentiation of the epididymis in fetal rats

Summary The fine structure of the Wolffian duct and alterations that occur with the differentiation of the epididymis have been studied in fetal rats ranging in length from 5 to 22 mm (12 to 19 days gestation). Contents of the columnar Wolffian duct cells include basal and perinuclear granular endoplasmic reticulum, sparse apical agranular endoplasmic reticulum, elongate mitochondria, and a small Golgi complex. Large membrane-bounded dense bodies contain cellular organelles and may represent parts of degenerating cells ingested by phagocytosis. During the development of the epididymis from the Wolffian duct in older fetuses, intracytoplasmic confronting cisternae of the granular endoplasmic reticulum become numerous, and the agranular reticulum in the apical part of the cell increases moderately in abundance. Concomitantly, the apical cell surface is modified. Many microvilli are formed, indentations of the plasma membrane appear between the microvilli, and the number of coated vesicles in the apical cytoplasm increases. These changes closely follow the onset of androgen secretion by the fetal testis, and it is suggested that they may occur in response to androgen. The relation of these differentiations to known adult functions of the epididymis is discussed.This study was supported by a research grant from the American Cancer Society (E-500), Program Project HD-02282 of the National Institutes of Health, and Health Sciences Advancement Award FR-02084 from the National Institutes of Health. The author is the recipient of a Research Career Development Award from the National Institute of General Medical Sciences. He wishes to acknowledge the technical assistance of Mrs. Heather Taylor.     

Androgen metabolism in tissue recombinants composed of adult urinary bladder epithelium and urogenital sinus mesenchyme

Epithelium of the adult mouse urinary bladder (BLE) was experimentally combined with mesenchyme of the urogenital sinus (UGM) and grown in intact male hosts to produce prostate-like glandular structures. To determine the extent to which the BLE is altered in a functional sense by inductive influences from UGM, investigations into the in vitro metabolism of tritiated testosterone (T) were undertaken. An isocratic high performance liquid chromatographic (HPLC) method was developed in order to separate the metabolites of T in mouse bladder, prostate and UGM + BLE tissue recombinants. Using a C-18 reversed phase column and a tetrahydrofuran (20): methanol (40): H2O (40) mobile phase, efficient and rapid separation of T, dihydrotestosterone, 3 alpha-androstanediol, androstenedione, androstanedione and androsterone was achieved. The identities of the radiolabeled T metabolites were confirmed by recrystallization to constant specific activity. The results of the present study revealed that tissue recombinants expressed testosterone metabolic profiles only partially toward that of the adult prostate. For example, percentage formation of 5 alpha-androstanedione, 3 alpha-androstanediol and unknown polar metabolites in the UGM + BLE resembled the prostate and differed significantly from the urinary bladder. Conversely, formation of the 3 beta-androstanediol and androsterone from testosterone resembled the urinary bladder and differed from the formation of these metabolites in the prostate. These results suggest that in contrast to histomorphology, androgen-induced DNA synthesis, androgen receptor binding activity and total tissue two-dimensional gel electrophoretic protein profiles, androgen metabolic profiles in the tissue recombinants showed only partial transformation into prostatic phenotypes. Analysis of steroid-metabolic profiles, therefore, may represent an exquisite and sensitive method to assess gene expression in various hormone-responsive target tissues.     

Fasciola hepatica: the ultrastructure of the epithelium of the seminal vesicle, the ejaculatory duct and the cirrus.

The ultrastructure of the seminal vesicle, ejaculatory duct, cirrus sac and cirrus is described. The epithelium of the seminal vesicle consists of a single layer of squamous to cuboidal cells. The apical ends of the cells have thin polymorphic lamellae and long narrow pits, both of which enclose normal spermatozoa. The cells have a moderate amount of GER and Golgi complexes which produce a lucid secretory body. The ejaculatory duct epithelium is composed of cuboidal to columnar cells between or through which project the terminal parts of the ducts of the unicellular prostate glands. The apical surfaces of the epithelia are extended into triangular or filiform projections having thin sinuous lamellae. The cytoplasm contains GER cisternae and Golgi complexes which synthesize a dense ovoid secretion. The cirrus sac and cirrus are covered by a thin modified tegument. The cirrus has many spines and the normal ratio of T1 and T2 type of secretory bodies, whereas the cirrus sac has few spines and the T2 type of secretory body predominates over the T1 type. The significance and possible functions of the structures observed in the three tissues are discussed.     

Cytologic features of seminal vesicle epithelium in aspiration biopsy smears of the prostate.

To determine the rate of occurrence of cells derived from seminal vesicles in routine needle aspirates of the prostate, the smears of 853 aspirates were reviewed. The cytologic features of cells originating from the seminal vesicles were described using air-dried smears stained by the May-Grünwald-Giemsa method. The differential diagnosis with malignancy is discussed.     

Specific protein synthesis in isolated epithelium of guinea-pig seminal vesicle. General features

Four intrinsic soluble proteins are synthesized and secreted by sexually mature guinea-pig seminal-vesicle mucosa, which comprises a monolayer of a homogeneous columnar epithelial cell. All four proteins can be extracted readily in 154mm-NaCl from the organ's luminal constituents in which they are present in high concentration. They are referred to as proteins 1, 2, 3 and 4 in order of their elution during DEAE-cellulose column chromatography. Specific primary antibodies were harvested from goats that had been inoculated with the purified vesicular proteins; secondary antibodies were obtained from a donkey inoculated with goat γ-globulins. Double-antibody-immunoprecipitation techniques were developed to precipitate the vesicular proteins. Thus proteins newly synthesized from ¹⁴C-labelled amino acids could be precipitated and the incorporated radioactivity assessed. Isolated seminal-vesicle mucosa, incubated in only a buffered salt solution containing glucose, readily synthesized the soluble secreted proteins from added [¹⁴C]lysine plus [¹⁴C]glycine, [¹⁴C]histidine plus [¹⁴C]glutamate, [¹⁴C]glutamine alone and [¹⁴C]arginine alone. The rates of incorporation (d.p.m./mg of total soluble protein) of labelled lysine and glycine and of labelled arginine were linear with time over 180min. With the other labelled precursors, rates diminished between 60 and 180min. Labelled protein could be detected after only 10–15min of incubation. Only 4–9% of the newly synthesized protein remained associated with the mucosa; the remainder was found in the cell-free incubation medium. The isolated seminal-vesicle mucosal preparation will provide a unique opportunity to study the synthesis and secretion of abundant cell-specific proteins by this androgen-dependent tissue.     

Epithelial-mesenchymal interactions in prostatic development. II. Biochemical observations of prostatic induction by urogenital sinus mesenchyme in epithelium of the adult rodent urinary bladder

Adult bladder epithelium (BLE) is induced to differentiate into glandular epithelium after association with urogenital sinus mesenchyme (UGM) and subsequent in vivo growth in syngeneic male hosts. Alteration of epithelial cytodifferentiation is associated with the expression of prostate-specific antigens, histochemical and steroid metabolic activities. These observations suggest that the inductive influence of the UGM has reprogrammed both the morphological and functional characteristics of the urothelium. In this report, differences regarding the mechanisms and effects of androgenic stimulation of prostate and bladder are exploited to determine the extent to which UGM plus BLE recombinants express a prostatelike, androgen-dependent phenotype. Results from cytosolic and autoradiographic binding studies suggest that androgen binding is induced in UGM plus BLE recombinants and that this activity is accounted for by the induced urothelial cells. In UGM plus BLE recombinants, androgen-induced [3H]thymidine or [35S]-methionine uptake analyzed by two-dimensional gel electrophoresis was qualitatively and quantitatively similar to that of prostate as opposed to bladder. These studies indicate that expression within BLE of prostatic phenotype is associated with a loss of urothelial characteristics and that androgen sensitivity is presumably a function of the inductive activities of the stroma.     

Androgens are necessary for the establishment of secretory protein expression in the guinea pig seminal vesicle epithelium.

The guinea pig seminal vesicle epithelium (GPSVE) synthesizes and secretes milligram quantities of four related secretory proteins in an androgen-dependent manner. To investigate the role of androgens in the establishment of secretory protein synthesis during the development of the GPSVE, animals were castrated at Day 5, approximately 10 days before secretory protein accumulation begins in intact animals. Castration did not eliminate secretory protein mRNA from the SVE, but it did indefinitely postpone the developmentally programmed increase in secretory protein mRNA. Injection of neonatally castrated guinea pigs with either estradiol or dexamethasone did not alter levels of secretory protein mRNAs. However, treatment of castrated neonates with either testosterone propionate or dihydrotestosterone (DHT) led to specific increases in secretory protein mRNAs within 4 days. Although neonatally castrated animals accumulated and translated significant amounts of secretory protein mRNA, the newly synthesized secretory proteins failed to accumulate until exogenous androgens were provided. This observation suggests that androgens regulate both the accumulation of secretory protein mRNA and the accumulation of secretory proteins in the GPSVE.     

Specific protein synthesis in isolated epithelium of guinea-pig seminal vesicle. Effects of castration and androgen replacement

Four intrinsic soluble secretory proteins are synthesized in vitro by isolated seminal-vesicle mucosa from sexually mature guinea pigs. Newly synthesized specific proteins labelled with [¹⁴C]glycine and [¹⁴C]lysine were precipitated by using double-antibody immunoprecipitation techniques and their radioactivity was assessed. Rates of synthesis were determined on each of 5 days after castration. By 5 days after castration the wet weight of the epithelium decreased to 42% of intact control values; the absolute amount of specific protein synthesized in vitro after 60min incubation decreased to 28% and the 27500g cytoplasmic protein content decreased to 31%. Thus androgen deprivation leads to a decrease in general protein synthesis in vivo, as well as to a decrease in specific protein synthesis in vitro. Specific protein synthesis comprised 76% of the total protein formed in isolated tissue from animals 5 days after castration as compared with 99–100% in tissue from intact animals. At 72h after an injection of testosterone or dihydrotestosterone, seminal-vesicle epithelium wet weight, cytoplasmic protein content and capability for synthesizing specific proteins in vitro were restored to approx. 70% of normal values. At 72h after onset of therapy with 3α-androstanediol, both epithelium wet weight and cytoplasmic protein content had increased significantly, but without a corresponding increase in the capability of the isolated tissue to synthesize specific proteins. The soluble labelled proteins synthesized in vitro by isolated epithelium from intact animals during 60 or 120min incubation were essentially entirely immunoprecipitable, i.e. specific. In contrast, approx. 29% of all soluble protein newly synthesized by isolated epithelium from animals 5 days after castration was acid-precipitable, but not immunoprecipitable, i.e. `non-specific'. The injection of testosterone into castrated animals inhibited the synthesis of the non-specific fraction by isolated tissue. The effects of castration on the ultrastructure of guinea-pig seminal-vesicle epithelium are also presented.     

Androgen-dependent synthesis of basic secretory proteins by the rat seminal vesicle.

1. Two basic proteins were purified from secretions of rat seminal vesicles by using Sephadex G-200 chromatography and polyacrylamide-gel electrophoresis under denaturing conditions. 2. It is not certain that these two proteins are distinct species and not subunits of a larger protein, but their properties are similar. Highly basic (pI = 9.7), they migrate to the cathode at high pH and their amino acid composition shows them to be rich in basic residues and serine. Threonine and hydrophobic residues are few. Both proteins are glycoproteins and have mol.wts. of 17000 and 18500. 3. Together these two proteins account for 25-30% of the protein synthesized by the vesicles, but they are absent from other tissues. 4. Changes in androgen status of the animal markedly affect these proteins. After castration, a progressive decrease in the basic proteins is observed and the synthesis of the two proteins as measured by [35S]methionine incorporation in vitro is is decreased. Testosterone administration in vivo rapidly restores their rates of synthesis. 5. These effects on specific protein synthesis are also observed for total cellular protein, and it is suggested that testosterone acts generally on the total protein-synthetic capacity of the cell and not specifically on individual proteins. Proliferative responses in the secretory epithelium may also be involved. 6. The extreme steroid specificity of the induction process suggests that the synthesis of these basic proteins is mediated by the androgen-receptor system. 7. The biological function of these proteins is not clear, but they do not appear to be involved in the formation of the copulatory plug.     

Induction of tubules in rat metanephrogenic mesenchyme in the absence of an inductive tissue

Differentiation of metanephrogenic mesenchyme to renal tubular epithelium requires induction by the ureteric bud in vivo or any of several embryonic tissues in vitro. In an effort to eliminate the tissue requirement in embryonic induction, extracellular matrices and soluble factors were analyzed individually or in combination for their ability to stimulate tubulogenesis in uninduced metanephrogenic mesenchyme from 13-gestation-day rat embryos. These evaluations have established that pituitary extract and epidermal growth factor (EGF) in concert with a matrix can promote morphogenesis of mesenchymal rudiments in culture. While type I collagen, laminin, or fibronectin matrices all promoted tubulogenesis in the presence of pituitary extract and EGF, type IV collagen proved the most effective. Under these conditions, tubules were induced in 23/24 mesenchymal rudiments by 9 days in culture. Mesenchyme was not induced prior to explanation since it formed no tubules when cultured in a medium that allowed tubulogenesis in intact embryonic kidneys. Preliminary characterization of the undefined factor in pituitary extract was consistent with a protein of molecular weight greater than 100,000 but less than 300,000. When uninduced metanephrogenic mesenchyme from mouse was used instead of rat tissue, a similar pattern of morphogenesis was not observed, suggesting that the described medium is inappropriate for promoting differentiation in mouse or, less likely, that different mechanisms mediate differentiation in rat and mouse. These studies show that embryonic induction can occur in explanted rat renal mesenchyme in an appropriate environment and does not require the presence of an inductive tissue.     

Epithelial-mesenchymal interactions in prostatic development. I. morphological observations of prostatic induction by urogenital sinus mesenchyme in epithelium of the adult rodent urinary bladder

Tissue recombinants of embryonic urogenital sinus mesenchyme (UGM) and epithelium of the urinary bladder (urothelium, BLE) of adult rats and mice were grown for 3-30 d in male syngeneic hosts. Short-term in vivo growth indicated that prostatic morphogenesis is initiated as focal outgrowths from the basal aspect of the adult urothelium. The solid epithelial buds elongate, branch, and subsequently canalize, forming prostatic acini. After 30 d of growth in the male hosts, prostatic acini exhibit secretory activity. The marked changes in urothelial morphology induced by the UGM are accompanied by the expression of fine- structural features indicative of secretory function (rough endoplasmic reticulum, Golgi apparatus, and secretory granules). During this process, urothelial cells express prostatic histochemical markers (alkaline phosphatase, nonspecific esterase, glycosaminoglycans) and prostate-specific antigens. The expression within BLE of prostatic characteristics is associated with the loss of urothelial characteristics. These data indicate that adult urothelial cells retain a responsiveness to embryonic mesenchymal inductors. Furthermore, mesenchyme-induced changes in urothelial cytodifferentiation appear to be coupled to changes in functional activity.     

Expression in male and genomic organization of the gene(s) coding for a major protein secreted by the rat seminal vesicle epithelium.

Double strand cDNA copies of lls poly(A)+mRNA purified from adult rat seminal vesicles (RSV), have been cloned in E.coli C600 using the Pst I site of pBR322. Filter hybridization, nucleotide sequence analysis and positive hybridization translation were used to demonstrate that one of the recombinant plasmids obtained (pRSV25) contained a 260 bp long insert coding for a significant part of the precursor to the protein IV present in the RSV secretion. By using labelled pRSV25 DNA we have found that high levels of RSV IV mRNA were present only in the rat seminal vesicle epithelium. The amounts of RSV IV mRNA present in other tissues of the same organism were below the levels detectable by the methods used. In addition, other data reported here indicate that the RSV IV gene(s) is present in both sexes, probably with a different organization.