Adult male mice conceived by in vitro fertilization exhibit increased glucocorticoid receptor expression in fat tissue.

Prenatal development is highly plastic and readily influenced by the environment. Adverse conditions have been shown to alter organ development and predispose offspring to chronic diseases, including diabetes and hypertension. Notably, it appears that the changes in glucocorticoid hormones or glucocorticoid receptor (GR) levels in peripheral tissues could play a role in the development of chronic diseases. We have previously demonstrated that in vitro fertilization (IVF) and preimplantation embryo culture is associated with growth alterations and glucose intolerance in mice. However, it is unknown if GR signaling is affected in adult IVF offspring. Here we show that GR expression is increased in inbred (C57Bl6/J) and outbred (CF-1× B6D2F1/J) blastocysts following in vitro culture and elevated levels are also present in the adipose tissue of adult male mice. Importantly, genes involved in lipolysis and triglyceride synthesis and responsive to GR were also increased in adipose tissue, indicating that increased GR activates downstream gene pathways. The promoter region of GR, previously reported to be epigenetically modified by perinatal manipulation, showed no changes in DNA methylation status. Our findings demonstrate that IVF results in a long-term change in GR gene expression in a sex- and tissue-specific manner. These changes in adipose tissues may well contribute to the metabolic phenotype in mice conceived by IVF.

In vitro culture of mouse embryos reduces differential gene expression between inner cell mass and trophectoderm.

Differences in gene expression and imprinting have been reported, comparing in vivo versus in vitro generated preimplantation embryos. Furthermore, mouse studies have shown that placenta development is altered following in vitro culture. However, the molecular mechanisms underlying these findings are unknown. We therefore isolated trophectoderm (TE) and inner cell mass (ICM) cells from in vivo and in vitro fertilization (IVF) embryos and evaluated their transcriptome using microarrays. We found that the transcriptomes of in vitro produced ICM and TE cells showed remarkably few differences compared to ICM and TE cells of in vivo generated embryos. In vitro fertilization embryos showed a reduced number of TE cells compared to in vivo embryos. In addition, TE of IVF embryos showed significant downregulation of solute transporter genes and of genes involved in placenta formation (Eomesodermin, Socs3) or implantation (Hbegf). In summary, IVF and embryo culture significantly affects the transcriptome of ICM and TE cells.

Effect of ICSI on gene expression and development of mouse preimplantation embryos.

BACKGROUND: In vitro culture (IVC) and IVF of preimplantation mouse embryos are associated with changes in gene expression. It is however not known whether ICSI has additional effects on the transcriptome of mouse blastocysts. METHODS: We compared gene expression and development of mouse blastocysts produced by ICSI and cultured in Whitten's medium (ICSI(WM)) or KSOM medium with amino acids (ICSI(KSOMaa)) with control blastocysts flushed out of the uterus on post coital Day 3.5 (in vivo). In addition, we compared gene expression in embryos generated by IVF or ICSI using WM. Global pattern of gene expression was assessed using the Affymetrix 430 2.0 chip. RESULTS: Blastocysts from ICSI fertilization have a reduction in the number of trophoblastic and inner cell mass cells compared with embryos generated in vivo. Approximately 1000 genes are differentially expressed between ICSI blastocyst and in vivo blastocysts; proliferation, apoptosis and morphogenetic pathways are the most common pathways altered after IVC. Unexpectedly, expression of only 41 genes was significantly different between embryo cultured in suboptimal conditions (WM) or optimal conditions (KSOM(aa)). CONCLUSIONS: Our results suggest that fertilization by ICSI may play a more important role in shaping the transcriptome of the developing mouse embryo than the culture media used.

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 Whitten's 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.

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.

Sex hormone-induced carcinogenesis in Rb-deficient prostate tissue.

The retinoblastoma (Rb) gene product is a prototypic tumor suppressor. Mice lacking the Rb gene are not viable and die in utero at approximately 13 days of gestation. In this study, we have rescued Rb-/- prostates by grafting pelvic organ rudiments from Rb-/- mouse embryos under the renal capsule of adult male nude mouse hosts. Grafts of embryonic pelvic organs developed into functional prostatic tissue. Some of the prostatic tissue generated was further used to construct chimeric prostatic tissue recombinants by combining wild-type rat urogenital mesenchyme (rUGM) with Rb-/- and Rb+/+ prostatic epithelium (PRE). The tissue recombinants were grown as subcapsular renal grafts and treated from the time of grafting with Silastic capsules containing 25 mg of testosterone plus 2.5 mg of estradiol. During 5-8 weeks of hormone treatment, rUGM+Rb+/+PRE tissue recombinants developed prostatic hyperplasia, whereas PRE in rUGM+Rb-/-PRE tissue recombinants developed hyperplasia, atypical hyperplasia, and carcinoma. During carcinogenesis in rUGM+Rb-/-PRE tissue recombinants, prostatic epithelial cells of the basal lineage disappeared, whereas the luminal cells underwent carcinogenesis. Epithelial E-cadherin almost totally disappeared. In all cases, epithelial PCNA labeling was elevated in tissue recombinants containing Rb-/- versus Rb+/+ epithelium. These epithelial changes were associated with almost total loss of smooth muscle cells in the stroma. In contrast, in untreated hosts rUGM+Rb+/+PRE tissue recombinants developed normally, and rUGM+Rb-/-PRE tissue recombinants developed mild epithelial hyperplasia. The results of this study demonstrate that Rb-/- prostatic tissue can be rescued from embryonic lethal mice and used to test its susceptibility to hormonal carcinogenesis. Deletion of the Rb gene predisposes prostatic epithelium to hyperplasia and increases proliferative activity Susceptibility to hormonal carcinogenesis in response to exogenous testosterone + estradiol is manifested in the progression from atypica hyperplasia to carcinoma. Thus, these findings demonstrate that the absence of the Rb tumor suppressor gene may predispose prostatic epithelial cells to carcinogenesis. Rescue of organs from Rb-/- embryos not only provides an opportunity to analyze the Rb gene pathway in the development and progression of prostate cancer but also provides an opportunity for specifically evaluating the role of the Rb pathway in development and carcinogenesis in other organs, such as the mammary gland and colon. Because rUGM greatly stimulates prostatic epithelial proliferation, the tissue recombinant model is a particularly useful tool for assessing the functional role of other genes in prostatic carcinogenesis through use of the appropriate transgenic or gene knockout mice.

Roles for Nkx3.1 in prostate development and cancer.

In aging men, the prostate gland becomes hyperproliferative and displays a propensity toward carcinoma. Although this hyperproliferative process has been proposed to represent an inappropriate reactivation of an embryonic differentiation program, the regulatory genes responsible for normal prostate development and function are largely undefined. Here we show that the murine Nkx3.1 homeobox gene is the earliest known marker of prostate epithelium during embryogenesis and is subsequently expressed at all stages of prostate differentiation in vivo as well as in tissue recombinants. A null mutation for Nkx3.1 obtained by targeted gene disruption results in defects in prostate ductal morphogenesis and secretory protein production. Notably, Nkx3.1 mutant mice display prostatic epithelial hyperplasia and dysplasia that increases in severity with age. This epithelial hyperplasia and dysplasia also occurs in heterozygous mice, indicating haploinsufficiency for this phenotype. Because human NKX3.1 is known to map to a prostate cancer hot spot, we propose that NKX3.1 is a prostate-specific tumor suppressor gene and that loss of a single allele may predispose to prostate carcinogenesis. The Nkx3.1 mutant mice provide a unique animal model for examining the relationship between normal prostate differentiation and early stages of prostate carcinogenesis.

Enlargement of the ampullary gland and seminal vesicle, but not the prostate in int-2/Fgf-3 transgenic mice.

Expression of the int2/Fgf-3 gene occurs during normal embryonic development and is associated with mammary cancer in mice. Overexpression of this gene under the control of the mouse mammary tumor virus long terminal repeat (MMTV-LTR) in males was reported to result in prostatic enlargement. In this report male Fgf-3-overexpressing mice were shown to have enlarged ampullary glands, seminal vesicles, and ductus deferens; there was extensive epithelial hyperplasia in the ampullary glands and seminal vesicles. The prostates of these animals were of normal size and histology. The transgene was expressed in all of the enlarged organs, which are derived exclusively from the Wolffian duct. Male secondary sex organs derived from the urogenital sinus, e.g., the ventral prostate, coagulating gland, and bulbourethral glands, were normal and did not express the MMTV-LTR-driven Fgf-3 transgene. A dorsolateral prostate was also morphologically normal but did express the transgene. This study underscores the importance of careful organ identification in transgenic models in which gross organ enlargement or distortion occurs. It also highlights the heterogeneity of the response to Fgf-3 among the secondary sex organs and even within the prostate itself.

Later onset of apoptosis in the bulbourethral glands after castration compared to that in the seminal vesicles.

Androgens affect many different target organs within the male reproductive tract to stimulate their development and secretory cytodifferentiation, and to maintain structure and function in adulthood. Castration causes regression of these organs via apoptosis. However, not all organs of the reproductive tract are equally sensitive to androgen withdrawal. The effects of castration on the mouse seminal vesicles (SVs) and bulbourethral glands (BUGs) were compared in terms of protein and DNA contents, epithelial apoptosis, and proliferative response of epithelial cells to androgen. Castration induced similar, marked decreases in protein contents in the SV and BUG by 2 days after castration which reached a minimum at 16 days post castration. Both organs underwent a decrease in DNA content, but the kinetics of this decline differed. In the SV, DNA content was significantly decreased by 4 days whereas in the BUG this did not occur until 16 days post castration. By day 16 both organs had regressed to roughly the same degree. The apoptotic index in the epithelium reflected this difference in timing as well. Apoptotic index of the SV epithelium was highest on day 3 after castration and declined thereafter. On the other hand, the apoptotic index in the BUG didn't begin to increase until 7 days after castration and became maximal on day 12. Daily injections of testosterone propionate (TP) from day 8, 16, or 30 after castration all increased epithelial labelling index in the SVs to a similar degree. However, the TP-induced increase in the epithelial labelling index in the BUG beginning on day 8 after castration was considerably less than that in BUGs receiving TP treatment from day 16 or 30 after castration. Thus, the proliferative response of the epithelium depended upon prior apoptosis in the gland, with the timing being delayed in the BUG as compared with the SV. The present results indicate that castration induces epithelial apoptosis and reduction in glandular DNA content considerably later in the BUG than in the SV though reduction in protein content in the BUG fell simultaneously with that in the SV.

Initiation of secretory activity of rat prostatic epithelium in organ culture.

Functional differentiation of prostatic epithelium is manifested by the production of tissue specific secretory proteins. In vivo production of these proteins is dependent on the presence of serum androgens. A serum-free organ culture system was used to examine the initiation of prostatic epithelial cytodifferentiation in vitro using two rat prostate specific secretory proteins (DP-1 and probasin) as markers of epithelial cytodifferentiation. The dorsal-lateral and anterior prostatic (AP) lobes from 12-day-old rats were cultured for 6 days in serum-free medium in the presence or absence of androgens. At the start of culture, secretory proteins DP-1 and probasin were undetectable using Western blot analysis. DP-1 and probasin were produced by explants cultured in the presence of androgens but were not detected in the absence of androgens. Dose-response studies were carried out for testosterone (T), 5 alpha-dihydrotestosterone (DHT), 5 alpha-Androstan-3 alpha, 17 beta-diol (3 alpha-Adiol), and two synthetic androgens: 17 alpha-methyl-19-nortestosterone (MENT) and methyltrienolone (R1881). All androgens used were capable of inducing expression of DP-1 and probasin in vitro. T, R1881, and Ment were effective at doses of 10(-7) M to 10(-9) M, whereas both DHT and 3 alpha-Adiol were able to induce DP-1 and probasin at concentrations as low as 10(-10) M. Estrogen (17 beta-Estradiol), hydrocortisone (11 beta, 17 alpha, 21-trihydroxypregn-4-ene-3, 20-dione), and progesterone (4-pregnen-3, 20-Dione) were ineffective in inducing prostatic secretory activity. Hydroxyflutamide (alpha-alpha-alpha-trifluro-2-methyl-4'-nitro-m-lactoluidide ) blocked the induction of secretory activity elicited by T. From histological sections, it was observed that explants cultured with T exhibited tall columnar epithelial morphology with organized stromal components. Tissue sections of explants cultured without T exhibited a cuboidal to low columnar morphology with less organized stromal components when compared with glands cultured with T. A DNA synthetic index was established to measure proliferation in the explants at the end of the culture period. Explants cultured in the presence of T exhibited greater DNA synthetic activity than explants cultured in the absence of T (P < 0.05). Using this serum-free model, we can explore the mechanism for the initiation of secretory cytodifferentiation.

Induction of prostatic morphology and secretion in urothelium by seminal vesicle mesenchyme.

Mesenchymal-epithelial interactions are essential for the development of the male reproductive tract. Tissue recombination experiments have been used to define the characteristics of these interactions. When mesenchyme, embryonic connective tissue, is recombined with epithelium from another organ an instructive induction may occur in which the developmental fate of the epithelium is altered. Instructive inductions are most common when the epithelium that is removed from the mesenchyme and the epithelium that is recombined with the mesenchyme are from the same germ layer. All of the mesenchyme of the male reproductive tract is of mesodermal origin. The epithelia of these organs are derived from either the mesodermal Wolffian duct epithelium or the endodermal urogenital sinus epithelium. Urogenital sinus mesenchyme can instructively induce bladder and urethral epithelium to form prostate (Donjacour, A. A. and Cunha, G. R. (1993) Endocrinol. 132, 2342-2350) and seminal vesicle mesenchyme can instructively induce epithelium from the ductus deferens and ureter (Cunha, G. R., Young, P., Higgins, S. J. and Cooke, P. S. (1991) Development 111, 145-158) to form seminal vesicle. To see whether inductive interactions could occur across germ layers in this system, seminal vesicle mesenchyme, normally associated with a mesodermal epithelium, was recombined with epithelium from neonatal or adult bladder or urethra, which are of endodermal origin. The resulting tissue recombinants were analyzed histologically and by immunocytochemistry and western blotting with antibodies to prostatic and seminal vesicle secretory proteins. Full prostatic differentiation was observed in tissue recombinants made with seminal vesicle mesenchyme plus either adult or neonatal bladder or urethral epithelium.(ABSTRACT TRUNCATED AT 250 WORDS)

Prostate cancer in a transgenic mouse.

Progress toward understanding the biology of prostate cancer has been slow due to the few animal research models available to study the spectrum of this uniquely human disease. To develop an animal model for prostate cancer, several lines of transgenic mice were generated by using the prostate-specific rat probasin promoter to derive expression of the simian virus 40 large tumor antigen-coding region. Mice expressing high levels of the transgene display progressive forms of prostatic disease that histologically resemble human prostate cancer, ranging from mild intraepithelial hyperplasia to large multinodular malignant neoplasia. Prostate tumors have been detected specifically in the prostate as early as 10 weeks of age. Immunohistochemical analysis of tumor tissue has demonstrated that dorsolateral prostate-specific secretory proteins were confined to well-differentiated ductal epithelial cells adjacent to, or within, the poorly differentiated tumor mass. Prostate tumors in the mice also display elevated levels of nuclear p53 and a decreased heterogeneous pattern of androgen-receptor expression, as observed in advanced human prostate cancer. The establishment of breeding lines of transgenic mice that reproducibly develop prostate cancer provides an animal model system to study the molecular basis of transformation of normal prostatic cells and the factors influencing the progression to metastatic prostate cancer.

Growth factors as mediators of androgen action during the development of the male urogenital tract.

Studies on the developing prostate and SV suggest that androgens act via mesenchymal AR to elicit synthesis and secretion of various autocrine and paracrine factors that regulate epithelial and stromal growth and differentiation. Clearly, the global regulation of epithelial growth and ductal branching morphogenesis is a complex multifactorial process involving the interplay of many diffusible factors (both positive and negative regulators), extracellular matrix molecules, cell-surface receptors for growth factors, receptors for extracellular matrix molecules, and matrix-degrading enzymes. Future progress will certainly be dependent upon the utilization of appropriate, biologically relevant models to examine the respective roles of various growth factors in the growth and development of androgen target organs.

Assessment of prostatic protein secretion in tissue recombinants made of urogenital sinus mesenchyme and urothelium from normal or androgen-insensitive mice.

Urogenital sinus mesenchyme (UGM) from normal, androgen receptor-positive mice appears to instructively induce prostatic morphology in urinary tract epithelium from both normal and androgen-insensitive (Tfm) mice. This indicates that epithelial androgen receptors are not necessary for prostatic development. However, the secretory function of these tissue recombinants has never been assessed. In this study antisera to androgen-dependent dorsolateral prostate (DLP) secretion were used in immunocytochemistry, Western blotting, and immunoprecipitation techniques to detect the presence of these prostatic proteins in tissue recombinants made with either normal or Tfm epithelium. In a majority of cases, UGM plus normal bladder or urethral epithelium developed into prostatic tissue that produced androgen-dependent DLP proteins. Hence UGM appeared to be capable of instructively inducing a functional prostatic phenotype in these normal heterotypic epithelia. With rare exceptions, tissue recombinants of UGM plus Tfm bladder or urethral epithelium did not produce full prostatic cytodifferentiation or mouse DLP proteins, indicating that epithelial androgen receptors are important for the final stages of morphogenesis and in the initiation of secretory function in the prostate.

Normal and abnormal development of the male urogenital tract. Role of androgens, mesenchymal-epithelial interactions, and growth factors.

Androgen-dependent male urogenital development occurs via mesenchymal-epithelial interactions in which mesenchyme induces epithelial morphogenesis, regulates epithelial proliferation, and evokes expression of tissue-specific secretory proteins. Mesenchymal-epithelial interactions continue to be important into adulthood. For example, mesenchyme of the urogenital sinus (UGM) and seminal vesicle (SVM) induce dramatic morphologic and functional changes in various adult epithelia. Since adult epithelial cells are unquestionably responsive to mesenchymes that can elicit expression of alternative morphologic and functional phenotypes, established carcinomas might also be influenced by their connective tissue environment. In this regard, Dunning prostatic tumor has been induced by UGM or SVM to differentiate into tall columnar secretory epithelial cells. This change in cytodifferentiation is associated with a reduction in growth rate and loss of tumorigenesis. The role of soluble growth factors in the mechanism of mesenchymal-epithelial interactions is discussed.

Androgen receptor expression and 5 alpha-reductase activity along the proximal-distal axis of the rat prostatic duct.

The rat prostate consists of a series of branched ducts that eminate from the urethra. Heterogeneity of rat prostatic growth, secretory activity, and cell turnover has been observed along the proximal-distal axis of the branched ductal network. In addition, there are regional differences in androgen sensitivity along the ducts, with the distal ductal tips being highly androgen dependent and the proximal regions being relatively androgen independent. To determine the underlying mechanisms that may regulate these regional differences in androgen responsiveness, androgen receptor (AR) levels and 5 alpha-reductase activity were examined along the proximal-distal axis of microdissected ventral prostatic ducts from 15-, 30-, and 100-day-old rats. As in the murine prostate, DNA synthetic activity was concentrated in the distal tip region of the 15- and 30-day ducts. Immunocytochemistry and autoradiography with [3H] dihydrotestosterone were used to examine AR expression and functional ability to bind ligand, respectively. The results revealed no discernable differences in AR levels or binding activity in any cell type along the ductal length in prepubertal, pubertal, or adult rats. In addition, 5 alpha-reductase activity was the same in the distal and proximal ductal regions. We conclude that regional heterogeneity in prostatic growth and function is not a result of differences in levels of AR and 5 alpha-reductase. Rather, other region-specific structural, intracellular, or paracrine factors may be responsible for the differences in androgen responsiveness along the prostatic duct.

Role of epithelial-mesenchymal interactions in the differentiation and spatial organization of visceral smooth muscle.

Uterine mesenchyme from newborn (0-day) rats was grown in association with epithelia from the adult cornea, urinary bladder, oesophagus, mammary gland, 1-day skin, and 1-day uterus. Following 1 month of growth, the differentiation of uterine mesenchyme into actin-positive smooth muscle cells was assessed immunocytochemically with antibodies to smooth muscle actin. Whereas grafts of uterine mesenchyme produced only small amounts of myometrium, all types of epithelia induced extensive myometrial differentiation in the uterine mesenchyme, which indicates that this effect is non-specific. The role of cell-cell interactions in the morphological patterning of smooth muscle layers was assessed by analysing tissue recombinants composed of adult prostatic epithelium (PRE) plus mesenchyme of the urogenital sinus (UGM), or seminal vesicle (SVM), or adult bladder epithelium (BLE) plus UGM or SVM. Prostatic ducts developed in all of these tissue recombinants (UGM + BLE, SVM + BLE, UGM + PRE and SVM + PRE). When UGM was used (UGM + PRE and UGM + BLE recombinants), actin-positive smooth muscle cells became organized into thin sheaths resembling the prostatic pattern. Conversely, when SVM was grown in association with PRE or BLE, the induced prostatic ducts were surrounded by thick layers of smooth muscle cells exhibiting the seminal vesicle pattern of organization. Smooth muscle cells were unorganized in grafts of SVM or UGM alone. These observations suggest that in male urogenital glands the mesenchyme dictates the spatial organization of the smooth muscle layers.(ABSTRACT TRUNCATED AT 250 WORDS)

Morphological and functional heterogeneity in the rat prostatic gland.

Ductal morphogenesis and adult ductal branching patterns were examined in the rat prostate by a microdissection method. The rat prostate consists of paired (right and left) subdivisions which correspond in large part to the classically defined lobes: ventral prostate, lateral prostate, dorsal prostate, and coagulating gland. Of particular interest was the finding that the lateral prostate consists of two different ductal zones: (1) lateral type 1 prostate with 5-7 long main ducts (resembling miniature palm trees) that extend cranially towards both the seminal vesicle and dorsal prostate to arborize near the bladder neck, and (2) lateral type 2 prostate with 5-6 short main ducts that arborize caudal to the bladder neck and give rise to compact bushy glands. Both lateral prostatic groups had a ductal-acinar organization. The adult structure of the other rat prostatic lobes was also examined, and closely resembled their mouse counterparts. The ventral prostate, which had 2-3 pairs of slender main ducts per side, and the coagulating gland, which had 1 main duct per side, was completely ductal in structure. In contrast, the dorsal prostate, which had 5-6 pairs of main ducts per side, had a ductal-acinar structure. Ductal branching morphogenesis occurred at different rates in different lobes and was essentially complete in the prostate at the 30 days. Immunocytochemical studies with an antibody to DP-1, a major secretory protein of the rat dorsal prostate, revealed that secretory function was initiated at approximately 30 days after birth in the coagulating gland, the dorsal prostate, and lateral type 1 prostate. A consistent feature of the lateral type 2 prostate was the absence of DP-1. On Western blots, DP-1 was detected in the secretion of the coagulating gland, lateral type 1 and dorsal prostate, but not in the ventral and lateral type 2 prostate. Polyacrylamide gel electrophoresis confirmed this result and demonstrated that the lateral type 2 prostate expressed several low-molecular weight secretory proteins not found in the other lobes of the prostate. On the whole, the rat prostate exhibited considerable heterogeneity both between and within lobes in developmental processes, ductal patterning, histology, and functional expression.

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.

Characterization of antibodies to androgen-dependent secretory proteins of the mouse dorsolateral prostate.

The mouse prostate is an attractive model for studying the relationship between epithelial-mesenchymal interactions and the mechanism of androgen action because of the volume of information on tissue interactions in the development of the prostate of this species and the existence of a mutant mouse lacking functional androgen receptors (Tfm mouse). In this paper the major proteins of the mouse dorsolateral prostate (DLP) have been described, and antibodies to these proteins have been characterized. The two most abundant secreted proteins were of 110,000-115,000 (Mj1) and 55,000-62,000 (Mj2) mol wt. They were glycosylated, androgen dependent, and appeared to exist in an oligomeric complex. Antibodies raised against mouse DLP secretion reacted mainly with Mj1, Mj2, and a minor protein of 140,000 mol wt (Mn1). The antibodies were of a high titer and recognized these three mouse DLP proteins by Western blotting, immunoprecipitation, and immunocytochemical techniques. Mj1 and Mj2 were antigenically similar to proteins in the mouse coagulating gland and in the rat DLP, but were not found in other organs. Immunocytochemical staining of the DLP from intact mice revealed many ducts that were lined by a tall columnar epithelium whose cells stained intensely. However, ducts that were distended with luminal secretion had a low columnar epithelium that rarely showed intracellular staining. These marker proteins and the antibodies to them will be useful for detecting androgen-dependent functional activity in tissue recombinant studies with a variety of experimental tissues.