Differential age-associated regulation of clusterin expression in prostate lobes of brown Norway rats.

BACKGROUND: Serum androgen concentrations decline with age in male Brown Norway rats and castration induces apoptosis of luminal secretory epithelial cells in the ventral but not in the dorsal and lateral prostate lobes. Clusterin has been described as an androgen-repressed gene and a protein with either anti- or pro-apoptotic actions. METHODS: We measured clusterin mRNA and protein levels, the effects of aging and castration on clusterin protein levels and clusterin immunolocalization within the prostatic ductal network in the prostate lobes of young and aged rats. RESULTS: Whereas levels of clusterin mRNA and protein expression measured by RT-PCR and Western blot, respectively, were higher in the ventral and lateral lobes of aged (24 months) compared to young (4 months) rats, no age-dependent differences were observed in the dorsal lobe. Clusterin expression was localized by immunohistochemistry exclusively to the proximal duct segment of young rats, but extended to the distal segment of the ventral and lateral lobes of aged rats. Despite an age-related decrease in serum testosterone concentration, clusterin gene expression was not altered in the dorsal lobe. After castration, levels of clusterin expression increased significantly in the ventral and lateral lobes despite the absence of epithelial cell apoptosis in the latter. In castrated rats, clusterin expression extended throughout the proximal-distal duct regions of the prostate lobes of young and aged rats. CONCLUSION: Regulation of clusterin expression in the prostate lobes of aging rats appears complex and is neither directly repressed by androgen nor dependent on apoptotic-induced stress.

Gene expression changes are age-dependent and lobe-specific in the brown Norway rat model of prostatic hyperplasia.

BACKGROUND: Benign prostatic hyperplasia (BPH) is an age-related enlargement of the prostate, characterized by increased proliferation of stromal and epithelial cells. Despite its prevalence, the etiology of BPH is unknown. METHODS: The Brown Norway rat is a model for age-dependent, lobe-specific hyperplasia of the prostate. Histological analyses of the dorsal and lateral lobes from aged rats reveal focal areas characterized by increased numbers of luminal epithelial cells, whereas the ventral lobe is unaffected. This study examined differential gene expression by lobe and age in the Brown Norway rat prostate. The objective was to identify genes with different levels of expression in the prostate lobes from 4-month (young) and 24-month (old) animals, and to subsequently link changes in gene expression to mechanisms of prostate aging. RESULTS: The number of age-dependent differentially expressed genes was greatest in the dorsal compared to the ventral and lateral lobes. Minimal redundancy was observed among the differentially expressed genes in the three lobes. Age-related changes in the expression levels of 14 candidate genes in the dorsal, lateral and ventral lobes were confirmed by quantitative RT-PCR. Genes that exhibited age-related differences in their expression were associated with proliferation, oxidative stress, and prostate cancer progression, including topoisomerase II alpha (Topo2a), aurora kinase B (Aurkb), stathmin 1 (Stmn1), and glutathione S-transferase pi. Immunohistochemistry for Topo2a, Aurkb, and Stmn1 confirmed age-related changes in protein localization in the lateral lobe of young and aged prostates. CONCLUSION: These findings provide clues to the molecular events associated with aging in the prostate.

Transgenerational epigenetic programming of the embryonic testis transcriptome.

Embryonic exposure to the endocrine disruptor vinclozolin during gonadal sex determination appears to promote an epigenetic reprogramming of the male germ line that is associated with transgenerational adult-onset disease states. Transgenerational effects on the embryonic day 16 (E16) testis demonstrated reproducible changes in the testis transcriptome for multiple generations (F1-F3). The expression of 196 genes was found to be influenced, with the majority of gene expression being decreased or silenced. Dramatic changes in the gene expression of methyltransferases during gonadal sex determination were observed in the F1 and F2 vinclozolin generation (E16) embryonic testis, but the majority returned to control-generation levels by the F3 generation. The most dramatic effects were on the germ-line-associated Dnmt3A and Dnmt3L isoforms. Observations demonstrate that an embryonic exposure to vinclozolin appears to promote an epigenetic reprogramming of the male germ line that correlates with transgenerational alterations in the testis transcriptome in subsequent generations.

Transforming growth factor beta (TGFbeta1, TGFbeta2 and TGFbeta3) null-mutant phenotypes in embryonic gonadal development.

The role transforming growth factor beta (TGFb) isoforms TGFb1, TGFb2 and TGFb3 have in the regulation of embryonic gonadal development was investigated with the use of null-mutant (i.e. knockout) mice for each of the TGFb isoforms. Late embryonic gonadal development was investigated because homozygote TGFb null-mutant mice generally die around birth, with some embryonic loss as well. In the testis, the TGFb1 null-mutant mice had a decrease in the number of germ cells at birth, postnatal day 0 (P0). In the testis, the TGFb2 null-mutant mice had a decrease in the number of seminiferous cords at embryonic day 15 (E15). In the ovary, the TGFb2 null-mutant mice had an increase in the number of germ cells at P0. TGFb isoforms appear to have a role in gonadal development, but interactions between the isoforms is speculated to compensate in the different TGFb isoform null-mutant mice.

Transgenerational epigenetic effects of the endocrine disruptor vinclozolin on pregnancies and female adult onset disease.

Endocrine disruptor exposure during gonadal sex determination was previously found to induce male rat adult onset transgenerational disease (F1-F4 generation), and this was associated with an alteration in the epigenetic (i.e., DNA methylation) programming of the male germ line. The current study was designed to characterize the transgenerational disease phenotypes of the female adult offspring. Pregnant rats (F0 generation) were treated transiently with vinclozolin (i.e., fungicide with anti-androgenic activity) on embryonic (E) days E8-E14 of gestation. F1 control and vinclozolin generation offspring from different litters were mated to produce F2 offspring, and similarly F2 generation animals produced F3 generation offspring. Observations demonstrated that 9 out of 105 pregnant rats (8.6%) from the vinclozolin F1-F3 generations exhibited uterine hemorrhage and/or anemia late in pregnancy. None (0 out of 82) of the control F1-F3 generation females had similar pregnancy problems. Complete blood cell counts and serum chemistry profiles demonstrated that selected vinclozolin generation animals, but not controls, exhibited marked regenerative anemia in late pregnancy. Examination of kidney histology revealed moderate or severe glomerular abnormalities in 67% of the vinclozolin F2 and F3 generation adult females compared with 18% of the controls. Adult female vinclozolin generation animals also developed various types of tumors in 6.5% of the animals (11 out of 170), while 2% of control-line animals (3 out of 151) developed mammary tumors. Observations demonstrate that vinclozolin exposure during gonadal sex determination promotes a transgenerational increase in pregnancy abnormalities and female adult onset disease states.

Comparative anti-androgenic actions of vinclozolin and flutamide on transgenerational adult onset disease and spermatogenesis.

Exposure of gestating female rats to the anti-androgenic endocrine disruptor vinclozolin has been shown to induce transgenerational adult onset disease phenotypes. The current study, was designed to compare the actions of vinclozolin to the known anti-androgenic compound flutamide. The gestating female rats were exposed to intraperitoneal injections during embryonic day 8-14 (E8-E14) to 100mg/kg/day vinclozolin or flutamide at either 5mg or 20mg/kg/day. As previously observed, vinclozolin induced a transgenerational testis phenotype of increased spermatogenic cell apoptosis and decreased epididymal sperm number. In contrast, the flutamide exposures resulted in a testis phenotype of increased spermatogenic cell apoptosis and decreased epididymal sperm numbers in the F1 generation only, and not the F2 and F3 generation adult males. Interestingly, some of the low dose (5mg/kg) flutamide F2 generation offspring developed spinal agenesis and supernummery development (polymelia) of limbs. Although the actions of vinclozolin and flutamide appear similar in the F1 generation males, the transgenerational effects of vinclozolin do not appear to be acting through the same anti-androgenic mechanism as flutamide.

Epigenetic transgenerational actions of endocrine disruptors.

Endocrine disruptors have recently been shown to promote an epigenetic transgenerational phenotype involving a number of disease states (e.g. male infertility). The anti-androgenic fungicide vinclozolin was found to act transiently at the time of embryonic sex determination to promote in the F1 generation a spermatogenic cell defect and subfertility in the male. When the animals were allowed to age up to 1 yr, a number of other disease states developed. This phenotype was transferred through the male germ line to all subsequent generations analyzed (F1-F4). The ability of an environmental factor (i.e. endocrine disruptor) to promote an epigenetic transgenerational phenotype impacts the potential hazards of environmental toxins, mechanisms of disease etiology, and evolutionary biology. The biological importance of the epigenetic actions of environmental agents is reviewed in the context of the primordial germ cell and development of epigenetic transgenerational phenotypes.

Endocrine disruptor vinclozolin induced epigenetic transgenerational adult-onset disease.

The fetal basis of adult disease is poorly understood on a molecular level and cannot be solely attributed to genetic mutations or a single etiology. Embryonic exposure to environmental compounds has been shown to promote various disease states or lesions in the first generation (F1). The current study used the endocrine disruptor vinclozolin (antiandrogenic compound) in a transient embryonic exposure at the time of gonadal sex determination in rats. Adult animals from the F1 generation and all subsequent generations examined (F1-F4) developed a number of disease states or tissue abnormalities including prostate disease, kidney disease, immune system abnormalities, testis abnormalities, and tumor development (e.g. breast). In addition, a number of blood abnormalities developed including hypercholesterolemia. The incidence or prevalence of these transgenerational disease states was high and consistent across all generations (F1-F4) and, based on data from a previous study, appears to be due in part to epigenetic alterations in the male germ line. The observations demonstrate that an environmental compound, endocrine disruptor, can induce transgenerational disease states or abnormalities, and this suggests a potential epigenetic etiology and molecular basis of adult onset disease.

Transgenerational epigenetic imprinting of the male germline by endocrine disruptor exposure during gonadal sex determination.

Embryonic exposure to the endocrine disruptor vinclozolin at the time of gonadal sex determination was previously found to promote transgenerational disease states. The actions of vinclozolin appear to be due to epigenetic alterations in the male germline that are transmitted to subsequent generations. Analysis of the transgenerational epigenetic effects on the male germline (i.e. sperm) identified 25 candidate DNA sequences with altered methylation patterns in the vinclozolin generation sperm. These sequences were identified and mapped to specific genes and noncoding DNA regions. Bisulfite sequencing was used to confirm the altered methylation pattern of 15 of the candidate DNA sequences. Alterations in the epigenetic pattern (i.e. methylation) of these genes/DNA sequences were found in the F2 and F3 generation germline. Therefore, the reprogramming of the male germline involves the induction of new imprinted-like genes/DNA sequences that acquire an apparent permanent DNA methylation pattern that is passed at least through the paternal allele. The expression pattern of several of the genes during embryonic development were found to be altered in the vinclozolin F1 and F2 generation testis. A number of the imprinted-like genes/DNA sequences identified are associated with epigenetic linked diseases. In summary, an endocrine disruptor exposure during embryonic gonadal sex determination was found to promote an alteration in the epigenetic (i.e. induction of imprinted-like genes/DNA sequences) programming of the male germline, and this is associated with the development of transgenerational disease states.

Transgenerational effect of the endocrine disruptor vinclozolin on male spermatogenesis.

The current study was designed to examine the actions of a model endocrine disruptor on embryonic testis development and male fertility. Pregnant rats (F0) that received a transient embryonic exposure to an environmental endocrine disruptor, vinclozolin, had male offspring (F1) with reduced spermatogenic capacity. The reduced spermatogenetic capacity observed in the F1 male offspring was transmitted to the subsequent generations (F2-F4). The administration of vinclozolin, an androgen receptor antagonist, at 100 mg/kg/day from embryonic day 8-14 (E8-E14) of pregnancy to only the F0 dam resulted in a transgenerational phenotype in the subsequent male offspring in the F1-F4 generations. The litter size and male/female sex ratios were similar in controls and the vinclozolin generations. The average testes/body weight index of the postnatal day 60 (P60) males was not significantly different in the vinclozolin-treated generations compared to the controls. However, the testicular spermatid number, as well as the epididymal sperm number and motility, were significantly reduced in the vinclozolin generations compared to the control animals. Postnatal day 20 (P20) testis from the vinclozolin F2 generation had no morphological abnormalities, but did have an increase in spermatogenic cell apoptosis. Although the P60 testis morphology was predominantly normal, the germ cell apoptosis was significantly increased in the testes cross sections of animals from the vinclozolin generations. The increase in apoptosis was stage-specific in the testis, with tubules at stages IX-XIV having the highest increase in apoptotic germ cells. The tubules at stages I-V also had an increase in apoptotic germ cells compared to the control samples, but tubules at stages VI-VIII had no increase in apoptotic germ cells. An outcross of a vinclozolin generation male with a wild-type female demonstrated that the reduced spermatogenic cell phenotype was transmitted through the male germ line. An outcross with a vinclozolin generation female with a wild-type male had no phenotype. A similar phenotype was observed in outbred Sprague Dawley and inbred Fisher rat strains. Observations demonstrate that a transient exposure at the time of male sex determination to the antiandrogenic endocrine disruptor vinclozolin can induce an apparent epigenetic transgenerational phenotype with reduced spermatogenic capacity.

Aging and the brown Norway rat leydig cell antioxidant defense system.

Previous studies have shown that testosterone production by the Leydig cells of aged Brown Norway rats is reduced from the relatively high levels produced by Leydig cells of young rats and that this reduction is not secondary to decreased serum luteinizing hormone concentration. The free radical theory of aging proposes that imbalance between pro-oxidants and the antioxidant defense system ultimately results in oxidative damage to cellular processes. With this in mind, we hypothesized herein that age-related reductions in steroidogenesis by Brown Norway rat Leydig cells may be associated with the impairment of the antioxidant defense system of these cells. To begin to test this hypothesis, we compared the activities and steady-state mRNA and protein levels of the antioxidant enzymes copper zinc (CuZn) superoxide dismutase (CuZnSOD, SOD1), manganese (Mn) superoxide dismutase (MnSOD, SOD2), and glutathione peroxidase (GPx) and the levels of reduced and oxidized glutathione in Leydig cells isolated from the testes of young (4-month-old) and aged (20-month-old) Brown Norway rats. For some studies, Leydig cells were isolated separately from aged testes that either had regressed because of age-related losses of germ cells or that were nonregressed. SOD (total) and GPx activities were found to decrease significantly with age whether or not the testes were regressed. CuZnSOD and MnSOD mRNA levels decreased with aging, though the magnitude of the decreases were considerably lower than the respective decreases in enzyme activities. GPx mRNA levels also decreased, which is consistent with the decreases seen in enzyme activity. MnSOD protein expression declined with age, and to a lesser extent, CuZnSOD did as well. Reduced and oxidized glutathione also exhibited age-related reductions in cells from both normal and regressed aged testes. The age-related decreases in Leydig cell antioxidant enzyme activities, gene expression, and protein levels and in glutathione were consistent with the hypothesis that the loss of steroidogenic function that accompanies Leydig cell aging may result in part from a decrease in the fidelity of the cellular antioxidant defense system.

Seminiferous cord formation and germ-cell programming: epigenetic transgenerational actions of endocrine disruptors.

The molecular and cellular control of embryonic testis development was investigated through an analysis of the embryonic testis transcriptome to identify potential regulatory factors for male sex determination and testis morphogenesis. One critical factor identified is neurotropin 3 (NT3). At the onset of male sex determination, Sertoli cells initiate differentiation and express NT3 to act as a chemotactic factor for mesonephros cells to migrate and associate with Sertoli-germ cell aggregates to promote cord formation. Promoter analysis suggests that NT3 may be an initial downstream gene to SRY and helps promote testis morphogenesis. Endocrine disruptors were used to potentially interfere with embryonic testis development and further investigate this biological process. The estrogenic pesticide methoxychlor and antiandrogenic fungicide vinclozolin were used. Previous studies have shown that methoxychlor and vinclozolin both interfere with embryonic testis cord formation and cause increased spermatogenic cell apoptosis in the adult testis. Interestingly, transient in vivo exposure to endocrine disruptors at the time of male sex determination caused a transgenerational phenotype (F1-F4) of spermatogenic cell apoptosis and subfertility. This apparent epigenetic mechanism involves altered DNA methylation and permanent re-programming of the male germ-line. A series of genes with altered DNA methylation and imprinting are being identified. Observations reviewed demonstrate that a transient embryonic in utero exposure to an endocrine disruptor influences the embryonic testis transcriptome and through epigenetic effects (e.g., DNA methylation) results in abnormal germ-cell differentiation that subsequently influences adult spermatogenic capacity and male fertility, and that this phenotype is transgenerational through the germ-line. The novel observations of transgenerational epigenetic endocrine disruptor actions on male reproduction critically impact the potential hazards of these compounds as environmental toxins. The literature reviewed provides insight into the molecular and cellular control of embryonic testis development, male sex determination, and the programming of the male germ-line.

Protein C inhibitor expression by adult rat Sertoli cells: effects of testosterone withdrawal and replacement.

Protein C inhibitor (PCI), a member of the plasma serine protease inhibitor family, has been reported to be abundantly expressed in the seminal vesicles and testes. In this study, we examine the localization and regulation of the PCI gene and protein expression in testes and freshly isolated Sertoli cells from control rats, rats treated with luteinizing hormone-suppressive testosterone/estradiol (TE)-containing Silastic capsules for 7, 14, 28, and 56 days, and rats treated with TE for 56 days, followed by high levels of testosterone for 7 or 14 days. The administration of the TE capsules for 56 days resulted in reduced testicular testosterone, from approximately 100 ng/mL in the controls to approximately 10 ng/mL, accompanied by a 73% reduction in testicular weight. PCI mRNA levels in freshly isolated Sertoli cells were reduced by 30% and 54% following TE treatment for 28 and 56 days, respectively. When rats that had received TE capsules for 56 days were provided replacement testosterone, there was a 40% increase in PCI mRNA levels within 7 days in the absence of any change in testicular weight, and PCI mRNA levels returned to control values by 14 days. The decrease in PCI mRNA levels in TE-treated rats was paralleled by a decrease in PCI protein levels in whole testis lysates and in seminiferous tubule fluid (STF). Protease activity was significantly increased in STF following 56 days of TE treatment. Taken together, these results indicate that 1) PCI in the testis is expressed by Sertoli cells; 2) the testicular expression of PCI is responsive to intratesticular testosterone levels; and 3) protease activity within the seminiferous epithelium is elevated when intratesticular concentration is decreased, perhaps as a consequence of decreased PCI.

Reduced intratesticular testosterone concentration alters the polymerization state of the Sertoli cell intermediate filament cytoskeleton by degradation of vimentin.

The Sertoli cell intermediate filament cytoskeleton is composed of the type III family member vimentin. The distribution of Sertoli cell vimentin varies with the stage of spermatogenesis, with shortening of the filaments at stages VII-VIII, the stages of spermiation. Experimental reduction in intratesticular testosterone (T) concentration also results in the sloughing of advanced spermatids from the Sertoli cells, as well as in the apoptotic death of spermatocytes. We hypothesized that alteration of the distribution of Sertoli cell vimentin might play a role in the loss of germ cells that occurs in response to reduced intratesticular T. To test this hypothesis, intratesticular T was reduced by implanting LH-suppressive SILASTIC brand capsules containing T and estradiol into adult rats for 8 wk. Immunohistochemical analyses revealed that, in response to the implants, the vimentin cytoskeleton collapsed around the Sertoli cell nuclei at all stages of the cycle, losing the extensive branching and structure normally seen at most stages of the cycle. Western blots of isolated Sertoli cells revealed that protein levels did not differ significantly between control and T- and estradiol-treated rats. However, Sertoli cell fractions containing the vimentin monomer revealed that vimentin was cleaved into four to five fragments in Sertoli cells in response to the implants, suggestive of proteolysis. These results indicate that, in response to reduced intratesticular T, the vimentin cytoskeleton of the Sertoli cell collapses to a perinuclear localization, and suggest that this collapse is associated with, and perhaps caused by, the degradation of the vimentin monomer rather than by loss of its expression.

An ex vivo analysis of Sertoli cell actin dynamics following gonadotropic hormone withdrawal.

The receptors for the steroid hormone testosterone and the peptide hormone follicle-stimulating hormone are localized to the somatic Sertoli cell in the seminiferous epithelium. In the rat, prolonged gonadotrophic hormone withdrawal has been shown to result in substantial germ cell apoptosis. Previous studies have shown that, coincident with the loss of germ cells following hypophysectomy, the actin cytoskeleton of the Sertoli cell becomes disorganized and diffuse throughout the cell's cytoplasm. The molecular mechanisms that govern Sertoli cell actin filament dynamics in response to the loss of gonadotrophic hormones remain undefined. It was therefore hypothesized that hypophysectomy brings about a decrease in the amount of polymerized actin (F-actin) within the Sertoli cell and that this decrease is associated with changes in the expression of genes known to govern Sertoli actin dynamics. To this end, Sertoli cells were isolated from adult control and hypophysectomized rats. Sertoli cells from hypophysectomized rats were found to contain significantly less (72%) F-actin relative to untreated controls, although overall, beta-actin protein and mRNA expression remained constant. The expression levels of genes known to directly influence the amount of F-actin in cells were then examined by Northern blot analysis. Cofilin and profilin I gene expression was unaffected by hypophysectomy, whereas the expression of profilin II and espin both decreased significantly (47% and 42%, respectively). Taken together, these results suggest that, following hypophysectomy, the actin cytoskeleton of the Sertoli cell shifts to a predominantly depolymerized state, perhaps in part because of decreases in profilin II and espin gene products.

Expression of testicular germ cell genes identified by differential display analysis.

Using differential display reverse transcriptase-polymerase chain reaction (DDRT-PCR) we identified transcripts encoding for the RNA helicase mDEAH9, Ran binding protein 5 (RanBP5), and 3 novel complementary DNAs designated GC3, GC12, and GC14 in developing testicular germ cells. Sources of RNA for the initial DDRT-PCR screen were purified mouse type A spermatogonia, adult mouse wild-type testis, and W/W(v) mutant mouse testis. We identified cDNA fragments for mDEAH9, RanBP5, GC3, GC12, and GC14 in testis and type A spermatogonia samples from wild-type mice, but not in samples from the W/W(v) mouse testis. These same transcripts were absent in Northern blots of testis RNA from mice treated with busulfan 30 days prior, but were present in testis RNA from wild-type mice at 5, 15, 25, and 40 days of age. The mDEAH9 gene was expressed in many tissues, whereas RanBP5 and GC12 genes were expressed predominantly in the testis with much lower expression in other tissues. The expression of GC3 and GC14 were limited to the testis as evidenced by Northern blot and RT-PCR analyses. The mDEAH9 transcript was not detected in cultured interstitial cells but was found at low levels in cultured immature Sertoli cells, whereas the RanBP5, GC3, GC12, and GC14 transcripts were not detected in either cultured testicular interstitial cells or cultured Sertoli cells. RT-PCR analyses of isolated spermatogonia, pachytene spermatocytes, and round spermatids revealed that mDEAH9, RanBP5, GC3, GC12, and GC14 genes were expressed in all 3 cellular populations. In situ hybridization analyses of testis samples from 40-day-old mice localized expression of mDEAH9, RanBP5, GC3, GC12, and GC14 to the seminiferous tubules. RanBP5 expression appeared to be regulated during the cycle of the seminiferous epithelium, with the highest expression in stages III through VII. Expression of GC14 was greatest in the meiotic germ cell populations.

Epigenetic transgenerational inheritance of vinclozolin induced mouse adult onset disease and associated sperm epigenome biomarkers.

The endocrine disruptor vinclozolin has previously been shown to promote epigenetic transgenerational inheritance of adult onset disease in the rat. The current study was designed to investigate the transgenerational actions of vinclozolin on the mouse. Transient exposure of the F0 generation gestating female during gonadal sex determination promoted transgenerational adult onset disease in F3 generation male and female mice, including spermatogenic cell defects, testicular abnormalities, prostate abnormalities, kidney abnormalities and polycystic ovarian disease. Pathology analysis demonstrated 75% of the vinclozolin lineage animals developed disease with 34% having two or more different disease states. Interestingly, the vinclozolin induced transgenerational disease was observed in the outbred CD-1 strain, but not the inbred 129 mouse strain. Analysis of the F3 generation sperm epigenome identified differential DNA methylation regions that can potentially be utilized as epigenetic biomarkers for transgenerational exposure and disease.

Alterations in the developing testis transcriptome following embryonic vinclozolin exposure.

The current study investigates the direct effects of in utero vinclozolin exposure on the developing F1 generation rat testis transcriptome. Previous studies have demonstrated that exposure to vinclozolin during embryonic gonadal sex determination induces epigenetic modifications of the germ line and transgenerational adult onset disease states. Microarray analyses were performed to compare control and vinclozolin treated testis transcriptomes at embryonic days 13, 14 and 16. A total of 576 differentially expressed genes were identified and the major cellular functions and pathways associated with these altered transcripts were examined. The sets of regulated genes at the different development periods were found to be transiently altered and distinct. Categorization by major known functions of altered genes was performed. Specific cellular process and pathway analyses suggest the involvement of Wnt and calcium signaling, vascular development and epigenetic mechanisms as potential mediators of the direct F1 generation actions of vinclozolin.

Epigenetic programming of the germ line: effects of endocrine disruptors on the development of transgenerational disease.

Epigenetic programming of the germ line occurs during embryonic development in a sex-specific manner. The male germ line becomes imprinted following sex determination. Environmental influences can alter this epigenetic programming and affect not only the developing offspring, but also potentially subsequent generations. Exposure to an endocrine disruptor (i.e. vinclozolin) during embryonic gonadal sex determination can alter the male germ-line epigenetics (e.g. DNA methylation). The epigenetic mechanism involves the alteration of DNA methylation in the germ line that appears to transmit transgenerational adult onset disease, including spermatogenic defects, prostate disease, kidney disease and cancer.

Transgenerational effects of the endocrine disruptor vinclozolin on the prostate transcriptome and adult onset disease.

PURPOSE: The ability of an endocrine disruptor exposure during gonadal sex determination to promote a transgenerational prostate disease phenotype was investigated in the current study. METHODS: Exposure of an F0 gestating female rat to the endocrine disruptor vinclozolin during F1 embryo gonadal sex determination promoted a transgenerational adult onset prostate disease phenotype. The prostate disease phenotype and physiological parameters were determined for males from F1 to F4 generations and the prostate transcriptome was assessed in the F3 generation. RESULTS: Although the prostate in prepubertal animals develops normally, abnormalities involving epithelial cell atrophy, glandular dysgenesis, prostatitis, and hyperplasia of the ventral prostate develop in older animals. The ventral prostate phenotype was transmitted for four generations (F1-F4). Analysis of the ventral prostate transcriptome demonstrated 954 genes had significantly altered expression between control and vinclozolin F3 generation animals. Analysis of isolated ventral prostate epithelial cells identified 259 genes with significantly altered expression between control and vinclozolin F3 generation animals. Characterization of regulated genes demonstrated several cellular pathways were influenced, including calcium and WNT. A number of genes identified have been shown to be associated with prostate disease and cancer, including beta-microseminoprotein (Msp) and tumor necrosis factor receptor superfamily 6 (Fadd). CONCLUSIONS: The ability of an endocrine disruptor to promote transgenerational prostate abnormalities appears to involve an epigenetic transgenerational alteration in the prostate transcriptome and male germ-line. Potential epigenetic transgenerational alteration of prostate gene expression by environmental compounds may be important to consider in the etiology of adult onset prostate disease.