Obesity induced by Borna disease virus in rats: key roles of hypothalamic fast-acting neurotransmitters and inflammatory infiltrates.

Human obesity epidemic is increasing worldwide with major adverse consequences on health. Among other possible causes, the hypothesis of an infectious contribution is worth it to be considered. Here, we report on an animal model of virus-induced obesity which might help to better understand underlying processes in human obesity. Eighty Wistar rats, between 30 and 60 days of age, were intracerebrally inoculated with Borna disease virus (BDV-1), a neurotropic negative-strand RNA virus infecting an unusually broad host spectrum including humans. Half of the rats developed fatal encephalitis, while the other half, after 3-4 months, continuously gained weight. At tripled weights, rats were sacrificed by trans-cardial fixative perfusion. Neuropathology revealed prevailing inflammatory infiltrates in the median eminence (ME), progressive degeneration of neurons of the paraventricular nucleus, the entorhinal cortex and the amygdala, and a strikingly high-grade involution of the hippocampus with hydrocephalus. Immune histology revealed that major BDV-1 antigens were preferentially present at glutamatergic receptor sites, while GABAergic areas remained free from BDV-1. Virus-induced suppression of the glutamatergic system caused GABAergic predominance. In the hypothalamus, this shifted the energy balance to the anabolic appetite-stimulating side governed by GABA, allowing for excessive fat accumulation in obese rats. Furthermore, inflammatory infiltrates in the ME and ventro-medial arcuate nucleus hindered free access of appetite-suppressing hormones leptin and insulin. The hormone transport system in hypothalamic areas outside the ME became blocked by excessively produced leptin, leading to leptin resistance. The resulting hyperleptinemic milieu combined with suppressed glutamatergic mechanisms was a characteristic feature of the found metabolic pathology. In conclusion, the study provided clear evidence that BDV-1 induced obesity in the rat model is the result of interdependent structural and functional metabolic changes. They can be explained by an immunologically induced hypothalamic microcirculation-defect, combined with a disturbance of neurotransmitter regulatory systems. The proposed mechanism may also have implications for human health. BDV-1 infection has been frequently found in depressive patients. Independently, comorbidity between depression and obesity has been reported, either. Future studies should address the exciting question of whether BDV-1 infection could be a link, whatsoever, between these two conditions.

Perinatal exposure to androgen excess and the effects on the rat uterine estradiol responsiveness.

Androgen exposure during sexual development induces alterations in steroidal target tissues. The objective of this study was to evaluate the uterine responsiveness to estradiol after perinatal androgenization. Pregnant Wistar rats were exposed to corn oil or testosterone propionate at 0.05, 0.1, or 0.2 mg/kg from gestational day 12 until postnatal day 21. Female offspring was challenged with estradiol (E2 ) after weaning (0.4 mg/kg) and at adulthood (10 or 100 µg/day), when the pituitary response was also evaluated. At adulthood, control and 0.05 mg/kg groups presented a uterine weight increment when exposed to 100 µg/day of E2 , 0.1 mg/kg group only responded to 10 µg/day of E2 , and the 0.2 mg/kg group showed increased uterine weight at both doses. The pituitary weight was similarly increased after estradiol stimulation in all experimental groups. In conclusion, testosterone propionate exposure induced an abnormal stimulation of uterine tissue growth by estrogen stimulus without affecting pituitary response. More studies are needed to clarify whether these alterations are capable of impairing the reproductive capacity of the female tract. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1460-1468, 2016.

Excess androgen during perinatal life alters steroid receptor expression, apoptosis, and cell proliferation in the uteri of the offspring.

Exposure to environmental chemicals may contribute to reproductive disorders, especially when it occurs in critical periods of development. The female reproductive system can be a target for androgens derived from environmental contaminants or pathological conditions. The purpose of this study was to assess the long-term effects of androgens on uterine tissue after maternal exposure limited to the time of gestation and lactation. Pregnant Wistar rats were treated with testosterone propionate (TP) at 0.05 mg/kg, 0.1 mg/kg, 0.2 mg/kg or corn oil (vehicle), s.c., from gestational day 12 until the end of lactation. The results show changes in the pattern of expression of receptors for estrogen, progesterone, and androgen at all doses tested, and decreases in both apoptosis and cell proliferation indices at 0.1 and 0.2 mg/kg. We conclude that early TP exposure, under these experimental conditions, causes changes in cellular and molecular parameters that are essential for normal uterine function in the adult.

Novel partners of SPAG11B isoform D in the human male reproductive tract.

Human sperm-associated antigen 11 (SPAG11) is closely related to beta-defensins in structure, expression, and function. Like the beta-defensins, SPAG11 proteins are predominantly expressed in the male reproductive tract, where their best-known major roles are in innate host defense and reproduction. Although several hypotheses have emerged to describe the evolution of beta-defensin and SPAG11 multifunctionality, few describe these multiple functions in terms of defensin interactions with specific proteins. To gain insight into the protein interaction potentials of SPAG11 and the signaling pathways that SPAG11 may influence, we used a yeast two-hybrid screening of a human testis-epididymis library. The results reveal human SPAG11B isoform D (SPAG11B/D) interactions with tryptase alpha/beta 1 (TPSAB1), tetraspanin 7 (TSPAN7), and attractin (ATRN). These interactions were confirmed by coimmunoprecipitation and glutathione S-transferase affinity matrix binding. SPAG11B/D and the three interacting proteins are expressed in the proximal epididymis, and all function in immunity and fertility pathways. We analyzed the functional consequences of SPAG11B/D interaction with TPSAB1 and showed that SPAG11B/D is both a substrate and a potent inhibitor of TPSAB1 activity. Furthermore, we show that (like SPAG11B/D) TSPAN7 and ATRN are associated with spermatozoa.

Effect of deprivation of endogenous follicle stimulating hormone on rat epididymis: a histological evaluation.

The growth and function of the epididymis are regulated by testosterone produced by Leydig cells in the testes. In the present study it was observed that neutralization of endogenous follicle stimulating hormone (FSH) in immature rats using a highly specific antiserum to ovine FSH resulted in changes in the histology of the epididymis along with a decrease (50-60%) in its weight compared with the normal serum-treated controls. These changes were observed in both rat and monkey epididymis without any decrease in serum testosterone, on which epididymis is known to be dependent. A detailed study was therefore carried out on the effects of deprivation of FSH or testosterone on the histology of epididymis. The changes in epididymal histology following FSH deprivation included a decrease in the size of the tubule lumen in the rat as well as in the adult male bonnet monkey in which the antiserum against ovine FSH was raised. Intensive vacuolization and uneven surface of the luminal epithelium was also observed. In contrast, the effect of deprivation of testosterone support by way of administration of LH antiserum or fiutamide resulted in a decrease in the size of the lumen and degenerative changes. These results suggest that cauda epididymidis is a target for FSH action.

Immunolocalization of alpha(1A)-adrenoceptors in rat and human epididymis.

Immunohistochemistry was conducted to analyze the cellular localization of alpha(1A)-adrenoceptors along rat and human epididymis. ADR-A, a polyclonal antibody that recognizes the specific C-terminal region of alpha(1A)-adrenoceptors, immunostained this adrenoceptor subtype in smooth muscle cells surrounding the epididymal tubules and interstitial blood vessels and in subpopulations of epithelial cells from adult rat and human caput and cauda epididymidis. The same cell types from rat epididymidis were immunostained by ADR-1, a polyclonal antibody that recognizes a common region of the three alpha(1)-adrenoceptor subtypes, alpha(1A), alpha(1B), and alpha(1D). Immunostaining with both antibodies was also conducted in adult rat and human vas deferens and seminal vesicle used as positive controls because of the abundance of alpha(1A)-adrenoceptors in these tissues. ADR-A- and ADR-1-positive immunostaining was differentially distributed depending on the antibody, method of tissue fixation (Bouin-fixed and fresh frozen tissues), species (rat and human), tissue (caput and cauda epididymidis), and age (immature and adult rats) analyzed. This is the first report immunolocalizing alpha(1A)-adrenoceptor along rat and human epididymis. The presence of this adrenoceptor subtype in epididymal smooth muscle and epithelial cells indicates their contribution to smooth muscle contractile responses and a possible role in the absorptive and/or secretory activities of the epithelium lining the epididymal duct. Taken together, our results should contribute to a better understanding of the physiological role of alpha(1)-adrenoceptors in the epididymidis and the importance of the sympathetic nervous system for male (in)fertility.

Cloning, expression and immunolocalization of alpha1-adrenoceptor in different tissues from rhesus monkey and human male reproductive tract.

This study reports the genomic organization of the rhesus alpha(1A)-adrenoceptor gene (ADRA1A). Full-length cloning of rhesus ADRA1A splice variants was achieved by combining PCR screening of a seminal vesicle cDNA library and 5'-RACE assays with total RNA from seminal vesicle. The classical ADRA1A mRNA (ADRA1A_v1) and six full-length ADRA1A splice variants were identified representing transcripts that code for functional (ADRA1A_v1, ADRA1A_v2a, ADRA1A_v3a, ADRA1A_v3d, ADRA1A_v3e) and truncated (ADRA1A_v2c and ADRA1A_v3c) receptor isoforms. Comparative analysis of the deduced amino acid sequence indicated that rhesus ADRA1A_i1 isoform (corresponding to the ADRA1A_v1 transcript) shares high identity to the amino acid sequence present in the classical alpha(1A)-adrenoceptor from human and other mammalian species. Partial nucleotide sequences for rhesus alpha(1B)-(ADRA1B) and alpha(1D)-adrenoceptor (ADRA1D) transcripts were also characterized. RT-PCR studies indicated differential distribution of all ADRA1A-related splice variants as well as ADRA1B and ADRA1D mRNAs, in tissues from rhesus and human male reproductive tract. Immunohistochemistry revealed alpha(1A)-adrenoceptor (ADRA1A_i1) immunostaining in smooth muscle cells and epithelial cells of rhesus efferent ductules, epididymis and seminal vesicle. Taken together the present results demonstrate that the complexity of the splicing mechanisms involved in the regulation of the ADRA1A gene is not restricted to human and is a common characteristic among Old World monkeys.

Characterization and functions of beta defensins in the epididymis.

The epididymal beta-defensins have evolved by repeated gene duplication and divergence to encode a family of proteins that provide direct protection against pathogens and also support the male reproductive tract in its primary function. Male tract defensins also facilitate recovery from pathogen attack. The beta-defensins possess ancient conserved sequence and structural features widespread in multi-cellular organisms, suggesting fundamental roles in species survival. Primate SPAG11, the functional fusion of two ancestrally independent beta-defensin genes, produces a large family of alternatively spliced transcripts that are expressed according to tissue-specific and species-specific constraints. The complexity of SPAG11 varies in different branches of mammalian evolution. Interactions of human SPAG11D with host proteins indicate involvement in multiple signaling pathways.

Novel aspects of the sperm-associated antigen 11 (SPAG11) gene organization and expression in cattle (Bos taurus).

Beta-defensins are small cationic peptides exhibiting broad spectrum antimicrobial properties. In humans, many beta-defensin genes are located within a cluster on chromosome 8p23. The sperm associated antigen 11 (SPAG11) gene is contained in this cluster and is unusual among the human beta-defensins due to its complex genomic structure and mRNA splicing pattern. Here we report the genomic organization of the Bos taurus SPAG11 gene located on chromosome 27q1.2, within a cluster of beta-defensin genes. The exon structures of the fused bovine SPAG11 gene and of the mosaic transcripts initiated at both A and B promoters were established, including identification of novel exons and transcripts not previously found in primate or rodent. Evolutionary analysis against primate, rodent, canine, and porcine orthologs was performed. In adult bulls SPAG11C, SPAG11E, and SPAG11U mRNAs were detected predominantly in the male reproductive tract, while SPAG11D transcript was detected in reproductive and nonreproductive tissues and SPAG11V and SPAG11W mRNAs were confined to testis. Differential expression of all six transcripts was observed in tissues from fetal and adult bulls, suggesting that similar mRNA splicing mechanisms govern SPAG11 gene expression during pre- and postnatal development. Immunolocalization of SPAG11C and SPAG11D/E was demonstrated in the epithelium of the epididymis and testis, and SPAG11D in association with epididymal spermatozoa. Recombinant full-length SPAG11D protein was strongly antibacterial, while the SPAG11E C-terminal peptide that contains the beta-defensin motif in its structure was somewhat less potent. Taken together, the results suggest that SPAG11 isoforms perform both immune and reproductive functions in cattle.

Identification, cloning and functional characterization of novel sperm associated antigen 11 (SPAG11) isoforms in the rat.

BACKGROUND: Sperm binding proteins and their C-terminal peptides of the Sperm Associated Antigen 11 (SPAG11) family were found to play an important role in epididymal innate immunity in addition to their role in sperm maturation. However, the expression of Spag11 transcripts in rodents is not well documented. METHODS: Computational analysis was employed to identify novel Spag11 isoforms in the rat. RT-PCR analyses were carried out on RNAs isolated from the male reproductive tract tissues of rat using gene specific primers for Spag11c and Spag11t. The identities of PCR products were confirmed by sequencing. Tissue distribution, developmental expression and androgen regulation of Spag11t and Spag11c were studied using RT-PCR. The antimicrobial activities of recombinant Spag11t and Spag11c were tested against E coli in a colony forming unit assay. RESULTS: In this study, we identified two novel Spag11 transcripts, namely, Spag11t and Spag11c derived from the long arm of chromosome 16 in the rat (Rattus norvegicus), using both in silico and molecular biology approaches. Spag11c is expressed in all three regions of the epididymis, in testis and in ovary but is absent from the seminal vesicle. Spag11t expression is confined to the caput and it is not expressed in the testis, seminal vesicle or ovary. Age dependent expression of Spag11t and Spag11c was observed in the epididymides of rats (10-60 day old). Their expression was found to be most abundant in the adult rat (60 day) suggesting roles in mature reproductive function. Further, both Spag11t and Spag11c expression was down regulated in castrated rat epididymides and the expression was maintained in the testosterone replaced castrated rats. SPAG11C is a potent antibacterial agent. SPAG11T also displayed bactericidal capacity although weaker than SPAG11C and SPAG11E. CONCLUSION: The abundant expression of Spag11t and Spag11c in the male reproductive tract suggests an important role in male reproductive tract immunity. Their expression is developmentally regulated and androgen dependent. Characterization of novel SPAG11 isoforms will contribute to our understanding of the role of epididymal proteins in sperm maturation and innate immunity.

Cells positive for microtubule-associated protein 1B (MAP 1B) are present along rat and human efferent ductules and epididymis.

Microtubule-associated protein 1B (MAP 1B) is a neuronal cytoskeleton marker with predominant expression in the developing nervous system. The present study provides evidence for the expression of this cytoskeleton protein in non-neuronal and neuronal cells along rat and human efferent ductules and epididymis (initial segment, caput, and cauda). Reverse transcription/polymerase chain reaction and Western blot analysis were used to confirm the presence of MAP 1B (mRNA and protein) in rat tissues. Immunohistochemical studies revealed MAP-1B-positive staining in columnar ciliated cells present in efferent ductules and in narrow cells located in the initial segment, in both rat and human. MAP-1B-positive basal cells, located underneath the columnar cells, were only identified in the initial segment and caput epididymidis of the rat. Qualitative analysis of tissues from 40-day-old and 120-day-old rats indicated that the number of MAP-1B-positive ciliated, narrow, and basal cells per tubule increased with sexual maturation. These immunoreactive cells did not stain for dopamine beta-hydroxylase or acetylcholinesterase, indicating that they were not adrenergic or cholinergic in nature. Immunohistochemical studies also revealed the presence of MAP-1B-positive staining in interstitial nerve fibers in caput and cauda epididymidis from both rat and human. Thus, the expression of MAP 1B is not confined to a specific cell type in rat and human efferent ductules and epididymis. The functional significance of this cytoskeleton protein in tissues from the male reproductive tract requires further investigation.

Microarray analysis of androgen-regulated gene expression in testis: the use of the androgen-binding protein (ABP)-transgenic mouse as a model.

BACKGROUND: Spermatogenesis is an androgen-dependent process, yet the molecular mechanisms of androgens' actions in testis are poorly understood. Transgenic mice overexpressing rat androgen-binding protein (ABP) in their testes have reduced levels of intratesticular androgens and, as a result, show a progressive impairment of spermatogenesis. We used this model to characterize changes in global gene expression in testis in response to reduced bioavailability of androgens. METHODS: Total RNA was extracted from testes of 30-day old transgenic and wild-type control mice, converted to cRNA, labeled with biotin, and hybridized to oligonucleotide microarrays. Microarray results were confirmed by real-time reverse transcription polymerase chain reaction. RESULTS: Three-hundred-eighty-one genes (3.05% of all transcripts represented on the chips) were up-regulated and 198 genes (1.59%) were down-regulated by at least a factor of 2 in the androgen-deficient animals compared to controls. Genes encoding membrane proteins, intracellular signaling molecules, enzymes, proteins participating in the immune response, and those involved in cytoskeleton organization were significantly overrepresented in the up-regulated group. Among the down-regulated transcripts, those coding for extracellular proteins were overrepresented most dramatically, followed by those related to proteolysis, cell adhesion, immune response, and growth factor, cytokine, and ion channel activities. Transcripts with the greatest potential impact on cellular activities included several transcription factors, intracellular signal transducers, secreted signaling molecules and enzymes, and various cell surface molecules. Major nodes in the up-regulated network were IL-6, AGT, MYC, and A2M, those in the down-regulated network were IL-2, -4, and -10, MAPK8, SOCS1, and CREB1. CONCLUSION: Microarray analysis followed by gene ontology profiling and connectivity analysis identified several functional groups of genes and individual genes responding to sustained reduction of androgen levels in the mouse testis. These include genes whose products function as transcription factors, cell surface molecules including ion channels, extra- and intracellular signaling molecules, and secreted enzymes with the potential of regulating cell-to-cell attachment. The transcription factors CREB1 (down-regulated) and MYC (up-regulated) may mediate the most important initial phases of the testicular response to reduced levels of androgens. These results suggest specific avenues for further research that will lead to a better understanding of how androgens regulate spermatogenesis.

Co-expression of vasopressin and androgen-binding protein in the rat hypothalamus.

In previous studies we have observed the expression of androgen binding protein (ABP) in the rat hypothalamo-neurohypophysial system. With immunocytochemical double staining we found partial co-localization with oxytocin. In the present study we used antibodies to the anti-diuretic hormone arginine vasopressin (AVP) for co-localization with ABP in the rat hypothalamus. Both antigens were seen in the magnocellular paraventricular and supraoptic nuclei. Dense fiber networks with varicosities containing both AVP and ABP immunoreactivity were visible throughout the hypothalamus, the median eminence and in the posterior pituitary lobe. Double immunostaining revealed also co-existence in the parvocellular portion of the paraventricular nucleus and in the suprachiasmatic nucleus. ABP immunoreactive neurons in the preoptic region were devoid of AVP staining, AVP neurons in the bed nucleus of the stria terminalis stained only occasionally for ABP. We conclude that both the magnocellular and the parvocellular hypothalamic vasopressin systems are capable of expressing the steroid binding globulin, which is probably subject to axonal transport, along with the peptide hormone. Intrahypothalamic expression of ABP may be among the mechanisms necessary for rapid actions of steroids on hypothalamic neuroendocrine systems.

Altered bioavailability of testosterone in androgen-binding protein-transgenic mice.

Serum and intra-testicular total and free testosterone levels in different age groups of mice (7-360-day-old) were analyzed by radioimmunoassay (RIA) in age-matched wild type (WT)-control and in transgenic mice homozygous to rat androgen-binding protein (ABP-TG), in order to identify possible causes of increased pre-pubertal germ cell apoptosis, spermatogenetic defect and reduced fertility seen in ABP-TG mice. Total intra-testicular testosterone levels in the pre-pubertal ABP-TG (7, 14, 21 and 30-day-old) mice were significantly lower than those in age-matched WT-controls. After puberty (60 days and older) the total intra-testicular testosterone levels were higher than those in age-matched WT-controls and increased gradually, peaking on day 180. Serum total testosterone levels in ABP-TG mice did not differ from those in WT-control until day 30. However, a significant increase in the level of serum total testosterone was observed from day 60. Serum and intra-testicular free testosterone levels were significantly lower in 30, 120, 180 and 360-day-old ABP-TG mice than in age-matched WT-controls. Immunohistochemistry for the cholesterol side-chain cleavage (cytochrome P450) enzyme and quantitative real-time RT-PCR analysis of mRNAs for androgen receptor and for enzymes related to steroidogenesis did not show any changes in 30-day-old ABP-TG mice, indicating that the rates of steroidogenesis and utilization were not altered. Human chorionic gonadotrophin (hCG) administration to adult ABP-TG mice increased the intra-testicular total and free testosterone as well as total germ cell counts. We conclude that the presence of greater than physiological concentration of ABP in the mouse testis alters the ratio of free/bound testosterone, and thereby decreases the availability of free testosterone. As a result, a heightened wave of germ cell apoptosis during the pre-pubertal period followed by a reduction in germ cell numbers and reduced fertility is seen in these mice.

Differential expression and antibacterial activity of epididymis protein 2 isoforms in the male reproductive tract of human and rhesus monkey (Macaca mulatta).

The epididymis protein 2 (EP2) gene, the fusion of two ancestral beta-defensin genes, is highly expressed in the epididymis and subject to species-specific regulation at the levels of promoter selection, transcription, and mRNA splicing. EP2 mRNA expression is also androgen dependent, and at least two of the secreted proteins bind spermatozoa. Alternative splicing produces more than 17 different EP2 mRNA variants. In this article, the expression of EP2 variants was profiled in different tissues from the human and rhesus monkey (Macaca mulatta) male reproductive tract using reverse transcriptase-polymerase chain reaction. Different EP2 mRNA variants were identified not only in human and rhesus testis and epididymis but also in the novel sites, seminal vesicle and prostate. Immunolocalization of EP2 protein in epithelial cells from rhesus and human seminal vesicle demonstrated that EP2 transcripts are translated in these tissues. In addition, two novel splicing variants, named EP2R and EP2S, were discovered. EP2C was the only splice variant expressed in all tissues tested from rhesus monkey. However, expression was not detected in human testis or seminal vesicle. For the first time, bactericidal function was demonstrated for EP2C, EP2K, and EP2L. Taken together, the results indicate that EP2 expression is more widespread in the male reproductive tract than realized previously. Whereas the activity of every EP2 variant tested thus far is antibacterial, further investigation may reveal additional physiological roles for EP2 peptides in the primate male reproductive tract.

Racial differences in prostate androgen levels in men with clinically localized prostate cancer.

PURPOSE: We sought to determine whether there are racial differences in androgenic stimulation within the prostate tissue microenvironment. MATERIALS AND METHODS: Steroid hormones were extracted from snap frozen tissue obtained intraoperatively from radical prostatectomy specimens of 36 black and 59 white Americans. Testosterone, dihydrotestosterone (DHT), androstenedione (ASD), dehydroepiandrosterone, dehydroepiandrosterone sulfate, sex hormone-binding globulin (SHBG) and prostate specific antigen (PSA) were measured using radioimmunoassay. The Wilcoxon 2 group test was performed to compare clinical characteristics and tissue steroid levels between white and black Americans. Nonparametric rank ANOVA was used to consider race and other clinical factors in a multivariable way. RESULTS: Black and white American men were similar with respect to serum PSA, and pathological grade and stage. However, black men were younger (p = 0.01) and had a significantly higher body mass index (p = 0.02). Black and white men had similar testosterone and DHT. However, black men had higher ASD (p = 0.006) and SHBG (p = 0.009). Racial differences in ASD (p = 0.015) and SHBG (p = 0.008) persisted after controlling for age, body mass index, PSA, and pathological Gleason sum and stage. CONCLUSIONS: Tissue levels of testosterone and DHT did not differ by race. However, black men had higher tissue ASD and SHBG than white men. Higher tissue ASD did not result in a greater conversion of ASD to testosterone in the prostate of black men. Higher tissue SHBG may activate the androgen receptor through cyclic adenosine monophosphate dependent pathways.

The androgen axis in recurrent prostate cancer.

PURPOSE: Prostate cancer that recurs during androgen deprivation therapy is referred to as androgen-independent. High levels of expression of androgen receptor and androgen receptor-regulated genes in recurrent prostate cancer suggest a role for androgen receptor and its ligands in prostate cancer recurrence. EXPERIMENTAL DESIGN: Recurrent prostate cancer specimens from 22 men whose prostate cancer recurred locally during androgen deprivation therapy and benign prostate specimens from 48 men who had received no prior treatment were studied. Androgen receptor expression was measured using monoclonal antibody and automated digital video image analysis. Tissue androgens were measured using radioimmunoassay. RESULTS: Epithelial nuclei androgen receptor immunostaining in recurrent prostate cancer (mean optical density, 0.284 +/- SD 0.115 and percentage positive nuclei, 83.7 +/- 11.6) was similar to benign prostate (mean optical density, 0.315 +/- 0.044 and percentage positive nuclei, 77.3 +/- 13.0). Tissue levels of testosterone were similar in recurrent prostate cancer (2.78 +/- 2.34 pmol/g tissue) and benign prostate (3.26 +/- 2.66 pmol/g tissue). Tissue levels of dihydrotestosterone, dehydroepiandrosterone, and androstenedione were lower (Wilcoxon, P = 0.0000068, 0.00093, and 0.0089, respectively) in recurrent prostate cancer than in benign prostate, and mean dihydrotestosterone levels, although reduced, remained 1.45 nM. Androgen receptor activation in recurrent prostate cancer was suggested by the androgen-regulated gene product, prostate-specific antigen, at 8.80 +/- 10.80 nmol/g tissue. CONCLUSIONS: Testosterone and dihydrotestosterone occur in recurrent prostate cancer tissue at levels sufficient to activate androgen receptor. Novel therapies for recurrent prostate cancer should target androgen receptor directly and prevent the formation of androgens within prostate cancer tissue.

Distribution of androgen-binding protein in the rat hypothalamo-neurohypophyseal system, co-localization with oxytocin.

Androgen-binding protein (ABP) is known to be expressed in the male and female rat hypothalamus. In the present study, we observed immunocytochemically ABP in neurons of the magnocellular hypothalamic nuclei, in the preoptic region and in the lateral hypothalamus. Dense fiber networks with varicosities, containing ABP immunofluorescence, were visible throughout the hypothalamus, the median eminence and in the posterior pituitary lobe. Double immunostaining revealed a partial coexistence of ABP-and oxytocin immunoreactivity in a portion of the magnocellular perikarya. ABP was isolated by affinity chromatography from hypothalamus homogenates. Western blots resulted in immunoreactive (IR) bands with an approximate molecular weight of 35 and 50 kDa. Mass spectrometry of these preparations confirmed the presence of ABP, which was almost identical to ABP isolated from rat testis. It is likely that ABP, expressed in magnocellular oxytocinergic neurons, is subject to axonal transport and release in the hypothalamo-neurohypophyseal system.

LCN6, a novel human epididymal lipocalin.

BACKGROUND: The lipocalin (LCN) family of structurally conserved hydrophobic ligand binding proteins is represented in all major taxonomic groups from prokaryotes to primates. The importance of lipocalins in reproduction and the similarity to known epididymal lipocalins prompted us to characterize the novel human epididymal LCN6. METHODS AND RESULTS: LCN6 cDNA was identified by database analysis in a comprehensive human library sequencing program. Macaca mulatta (rhesus monkey) cDNA was obtained from an epididymis cDNA library and is 93% homologous to the human. The gene is located on chromosome 9q34 adjacent LCN8 and LCN5. LCN6 amino acid sequence is most closely related to LCN5, but the LCN6 beta-barrel structure is best modeled on mouse major urinary protein 1, a pheromone binding protein. Northern blot analysis of RNAs isolated from 25 human tissues revealed predominant expression of a 1.0 kb mRNA in the epididymis. No other transcript was detected except for weak expression of a larger hybridizing mRNA in urinary bladder. Northern hybridization analysis of LCN6 mRNA expression in sham-operated, castrated and testosterone replaced rhesus monkeys suggests mRNA levels are little affected 6 days after castration. Immunohistochemical staining revealed that LCN6 protein is abundant in the caput epithelium and lumen. Immunofluorescent staining of human spermatozoa shows LCN6 located on the head and tail of spermatozoa with the highest concentration of LCN6 on the post-acrosomal region of the head, where it appeared aggregated into large patches. CONCLUSIONS: LCN6 is a novel lipocalin closely related to Lcn5 and Lcn8 and these three genes are likely products of gene duplication events that predate rodent-primate divergence. Predominant expression in the epididymis and location on sperm surface are consistent with a role for LCN6 in male fertility.

Dynamics of testicular germ cell apoptosis in normal mice and transgenic mice overexpressing rat androgen-binding protein.

The number and type of testicular germ cells undergoing apoptosis in different age groups of mice (from 7 to 360 days of age) was determined and compared in age-matched wild type (WT) control and in a transgenic (TG) mice homozygous to rat androgen binding protein (ABP) using flow cytometry. Flow cytometric quantification revealed that the total number of germ cells undergoing apoptosis did not differ significantly in WT and TG mice up to Day 14. From Day 21 to Day 60, the number of germ cells undergoing apoptosis was consistently higher in TG than in WT mice. Starting from Day 90, the number of germ cells undergoing apoptosis in TG mice was lower than controls until Day 360. In 21-60 days old TG mice, spermatogonia, S-Phase cells, and primary spermatocytes are the cell types undergoing apoptosis at significantly greater numbers than those in WT mice. However, starting from day 60, the total number of spermatids undergoing apoptosis was significantly lower in TG mice than in age-matched WT controls. TdT-mediated dUTP-biotin nick end labeling (TUNEL) in testicular sections from TG mice of 21 and 30 days of age confirmed the presence of increased numbers of apoptotic germ cells compared to their age matched controls. These data indicate that the continuous presence of greater than physiological concentrations of ABP in the mouse testis has a biphasic effect on the frequency of apoptosis in germ cells. The initial pre-pubertal increase in testicular germ cell apoptosis may result from direct or indirect actions of ABP and is likely to determine the subsequent life-death balance of germ cell populations in TG mice, whereas the subsequent reduction may result from maturation depletion. A wave of apoptosis during the pre-pubertal period is required for normal spermatogenesis to develop, and our data indicate that this apoptotic wave may be regulated by ABP and/or androgens.