A novel defensive mechanism against acetaminophen toxicity in the mouse lateral nasal gland: role of CYP2A5-mediated regulation of testosterone homeostasis and salivary androgen-binding protein expression.

To identify novel factors or mechanisms that are important for the resistance of tissues to chemical toxicity, we have determined the mechanisms underlying the previously observed increases in resistance to acetaminophen (APAP) toxicity in the lateral nasal gland (LNG) of the male Cyp2g1-null/Cyp2a5-low mouse. Initial studies established that Cyp2a5-null mice, but not a newly generated strain of Cyp2g1-null mice, were resistant to APAP toxicity in the LNG; therefore, subsequent studies were focused on the Cyp2a5-null mice. Compared with the wild-type (WT) male mouse, the Cyp2a5-null male mouse had intact capability to metabolize APAP to reactive intermediates in the LNG, as well as unaltered circulating levels of APAP, APAP-GSH, APAP-glucuronide, and APAP-sulfate. However, it displayed reduced tissue levels of APAP and APAP-GSH and increased tissue levels of testosterone and salivary androgen-binding protein (ABP) in the LNG. Furthermore, we found that ABP was able to compete with GSH and cellular proteins for adduction with reactive metabolites of APAP in vitro. The amounts of APAP-ABP adducts formed in vivo were greater, whereas the amounts of APAP adducts formed with other cellular proteins were substantially lower, in the LNG of APAP-treated male Cyp2a5-null mice compared with the LNG of APAP-treated male WT mice. We propose that through its critical role in testosterone metabolism, CYP2A5 regulates 1) the bioavailability of APAP and APAP-GSH (presumably through modulation of the rates of xenobiotic excretion from the LNG) and 2) the expression of ABP, which can quench reactive APAP metabolites and thereby spare critical cellular proteins from inactivation.

Hedgehog pathway activity in the LADY prostate tumor model.

BACKGROUND: Robust Hedgehog (Hh) signaling has been implicated as a common feature of human prostate cancer and an important stimulus of tumor growth. The role of Hh signaling has been studied in several xenograft tumor models, however, the role of Hh in tumor development in a transgenic prostate cancer model has never been examined. RESULTS: We analyzed expression of Hh pathway components and conserved Hh target genes along with progenitor cell markers and selected markers of epithelial differentiation during tumor development in the LADY transgenic mouse model. Tumor development was associated with a selective increase in Ihh expression. In contrast Shh expression was decreased. Expression of the Hh target Patched (Ptc) was significantly decreased while Gli1 expression was not significantly altered. A survey of other relevant genes revealed significant increases in expression of Notch-1 and Nestin together with decreased expression of HNF3a/FoxA1, NPDC-1 and probasin. CONCLUSION: Our study shows no evidence for a generalized increase in Hh signaling during tumor development in the LADY mouse. It does reveal a selective increase in Ihh expression that is associated with increased expression of progenitor cell markers and decreased expression of terminal differentiation markers. These data suggest that Ihh expression may be a feature of a progenitor cell population that is involved in tumor development.

Reduction of Raf-1 kinase inhibitor protein expression correlates with breast cancer metastasis.

PURPOSE: Raf-1 kinase inhibitor protein (RKIP) was originally identified as the first physiologic inhibitor of the Raf/mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (ERK) pathway. This pathway regulates fundamental cellular functions, including those that are subverted in cancer cells, such as proliferation, transformation, survival, and metastasis. Recently, RKIP has been recognized as a strong candidate for a metastasis suppressor gene in cell and animal model systems. Therefore, we investigated whether RKIP expression is altered in clinical specimens of human primary breast cancers and their lymph node metastases. EXPERIMENTAL DESIGN: Paraffin-embedded tumor samples from 103 breast cancer patients were examined immunohistochemically for the expression of RKIP, activated ERK, and apoptosis. The specificity of the antibodies used was validated by competition experiments with purified recombinant RKIP protein. RESULTS: RKIP expression was high in breast duct epithelia and retained to varying degrees in primary breast tumors. However, in lymph node metastases, RKIP expression was highly significantly reduced or lost (P = 0.000003). No significant correlations were observed between RKIP expression and histologic type, tumor differentiation grade, size, or estrogen receptor status. CONCLUSION: This is the first study of RKIP expression in a large clinical cohort. It confirms the results of cell culture and animal studies, suggesting that in human breast cancer, RKIP is a metastasis suppressor gene whose expression must be down-regulated for metastases to develop. RKIP expression is independent of other markers for breast cancer progression and prognosis.

p53-independent apoptosis during mammary tumor progression in C3(1)/SV40 large T antigen transgenic mice: suppression of apoptosis during the transition from preneoplasia to carcinoma.

Alterations in apoptosis and associated mechanisms during mammary tumor progression were investigated in transgenic mice expressing the SV40 large T antigen (T(AG)) driven by the rat prostatic steroid-binding protein C3(1) 5'-flanking region. Apoptosis levels, assessed by in situ end labeling, were low in normal mammary epithelial cells, highest in atypical hyperplasias (preneoplastic lesions), and less pronounced in adenocarcinomas. Preneoplastic cells maintain the ability to undergo apoptosis as a mechanism of tumor growth suppression, but this critical control of apoptosis is lost as these lesions progress to carcinomas. These alterations in apoptosis occur during mammary tumor progression in mice containing wild-type p53+/+ genotype as well as in mice with the p53-/- genotype. Thus, apoptosis in this tumor model occurs through a p53-independent mechanism. Because other studies have demonstrated p53-dependent apoptosis in T(AG)-induced choroid plexus tumors of transgenic mice, we propose that the role of p53 in apoptosis may be tissue-specific. In addition, bcl-2 protein was not expressed in any mammary lesions. SV40 T(AG) expression, which correlated with the nuclear p53 protein at all stages of tumor progression, was low in normal mammary epithelial cells, moderately high in atypical hyperplasias, and strongly expressed in adenocarcinomas. No p53 mutations were found at any stage of mammary adenocarcinoma development, suggesting that tumor progression does not require a dominantly acting p53 mutation in this transgenic model. p2l(Waf1/Cip1), a cyclin-dependent kinase inhibitor, was expressed in normal mammary tissue but was not detected in the mammary carcinomas, despite high nuclear accumulation of wild-type p53 protein, suggesting functional loss of p53 due to binding of SV40 T(AG), to p53. These findings suggest that suppression of apoptosis during the transition from atypical hyperplasia to adenocarcinoma appears to be a critical event for mammary cancer development in C3(1)/T(AG) transgenic mice and occurs by p53- and bcl-2-independent pathways.

Inhibition of the Raf-MEK1/2-ERK1/2 signaling pathway, Bcl-xL down-regulation, and chemosensitization of non-Hodgkin's lymphoma B cells by Rituximab.

Rituximab (Rituxan, IDEC-C2B8) has been shown to sensitize non-Hodgkin's lymphoma (NHL) cell lines to chemotherapeutic drug-induced apoptosis. Rituximab treatment of Bcl-2-deficient Ramos cells and Bcl-2-expressing Daudi cells selectively decreases Bcl-(xL) expression and sensitizes the cells to paclitaxel-induced apoptosis. This study delineates the signaling pathway involved in rituximab-mediated Bcl-(xL) down-regulation in Ramos and Daudi NHL B cells. We hypothesized that rituximab may interfere with the extracellular signal-regulated kinase (ERK) 1/2 pathway, leading to decreased Bcl-(xL) expression. Rituximab (20 microg/mL) inhibited the kinase activity of mitogen-activated protein kinase kinase (MEK) 1/2 and reduced the phosphorylation of the components of the ERK1/2 pathway (Raf-1, MEK1/2, and ERK1/2) and decreased activator protein-1 DNA binding activity and Bcl-(xL) gene expression. These events occurred with similar kinetics and were observed 3 to 6 hours after rituximab treatment. Rituximab-mediated effects were corroborated by using specific inhibitors of the ERK1/2 pathway, which also reduced Bcl-(xL) levels and sensitized the NHL B cells to paclitaxel-induced apoptosis. Previous findings implicated a negative regulatory role of the Raf-1 kinase inhibitor protein (RKIP) on the ERK1/2 pathway. Rituximab treatment of NHL B cells significantly up-regulated RKIP expression, thus interrupting the ERK1/2 signaling pathway through the physical association between Raf-1 and RKIP, which was concomitant with Bcl-(xL) down-regulation. These novel findings reveal a signaling pathway triggered by rituximab, whereby rituximab-mediated up-regulation of RKIP adversely regulates the activity of the ERK1/2 pathway, Bcl-(xL) expression, and subsequent chemosensitization of drug-refractory NHL B cells. The significance of these findings is discussed.

[Effects of p,p'-dDE on the expression of androgen-binding protein, transferrin and inhibin B mRNA in rat sertoli cells].

OBJECTIVE: To explore effects of p, p'DDE on the expression of androgen binding protein (ABP), transferrin (Tf) and inhibin B (INH B) mRNA in testis Sertoli cells of Sprague Dawley rats. METHODS: A method has been set up to obtain a large number of viable Sertoli cells from SD rats of 18-20 days of age. With a series of concentration p,p'-DDE (10, 30, and 50 micromol/L) co-incubating the Sertoli cells in vitro, the expression of ABP, Tf and INH B mRNA were determined by RT-PCR. RESULTS: a) With increase of the incubated p, p'-DDE, the expression of ABP mRNA in Sertoli cells went up while that of Tf and INH B dropped in a dose-dependent manner (P < 0. 05). b) The correlation analysis among ABP, Tf and INH B showed that negative relationships were found between ABP and Tf or INH B, respectively (r = - 0. 391 3, P = 0. 032 5; r = - 0.235 2, P = 0.0158), and that positive correlation was indicated between Tf and INH B (r =0.4516, P =0.0047). CONCLUSION: p,p'-DDE is a reproductive toxicant which disrupts the transcription of ABP, Tf and INH B in rat Sertoli cells so as to result in reproductive dysfunction.

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.

The rat probasin gene promoter directs hormonally and developmentally regulated expression of a heterologous gene specifically to the prostate in transgenic mice.

An expression cassette carrying 426 basepairs of the rat probasin (PB) gene promoter and 28 basepairs of 5'-untranslated region is sufficient to target the expression of the bacterial chloramphenicol acetyltransferase (CAT) gene specifically to the prostate in transgenic mice. The PB-CAT transgene was expressed in three of five (60%) independent lines of mice, and this expression, as reported previously for the endogenous rat gene, was male specific, restricted primarily to the lateral, dorsal, and ventral lobes of the prostate, with only very low levels of CAT activity detected in the anterior prostate and seminal vesicles. The developmental and hormonal regulation of the transgene also paralleled that reported for the rat gene, with a 70-fold increase in CAT activity in the mouse prostate observed between 2-7 weeks of age, a time corresponding to sexual maturation. PB-CAT activity in the prostate declined after castration to 3.5% of the precastration level, and the CAT activity in castrated males approached precastration levels when mice were supplemented with testosterone. Transgene expression in castrated males was not induced by dexamethasone. Coinjection of PB-CAT with a chicken lysozyme gene matrix attachment region resulted in their cointegration and further restricted the pattern of PB-CAT to the dorsolateral prostate, with suppressed expression observed in the ventral prostate. These studies demonstrate that a minimal rat probasin promoter can target heterologous gene expression specifically to the prostate in a developmentally and hormonally regulated fashion.

Tissue specificity of the hormonal response in sex accessory tissues is associated with nuclear matrix protein patterns.

The DNA of interphase nuclei have very specific three-dimensional organizations that are different in different cell types, and it is possible that this varying DNA organization is responsible for the tissue specificity of gene expression. The nuclear matrix organizes the three-dimensional structure of the DNA and is believed to be involved in the control of gene expression. This study compares the nuclear structural proteins between two sex accessory tissues in the same animal responding to the same androgen stimulation by the differential expression of major tissue-specific secretory proteins. We demonstrate here that the nuclear matrix is tissue specific in the rat ventral prostate and seminal vesicle, and undergoes characteristic alterations in its protein composition upon androgen withdrawal. Three types of nuclear matrix proteins were observed: 1) nuclear matrix proteins that are different and tissue specific in the rat ventral prostate and seminal vesicle, 2) a set of nuclear matrix proteins that either appear or disappear upon androgen withdrawal, and 3) a set of proteins that are common to both the ventral prostate and seminal vesicle and do not change with the hormonal state of the animal. Since the nuclear matrix is known to bind androgen receptors in a tissue- and steroid-specific manner, we propose that the tissue specificity of the nuclear matrix arranges the DNA in a unique conformation, which may be involved in the specific interaction of transcription factors with DNA sequences, resulting in tissue-specific patterns of secretory protein expression.

Regulation of gene expression in rat prostate by androgen and beta-adrenergic receptor pathways.

Denervation of rat ventral prostate has been accomplished by excising prostatic tissue fragments and implanting them under the renal capsules of intact syngeneic rats. This resulted in a substantial reduction of expression of a major organ-specific secretory protein, prostatic binding protein (PBP). The depressed level of PBP and its subunits and mRNAs could be restored, however, to as much as 80% of control levels by the administration of a pharmacological dose of exogenous androgen, testosterone propionate (TP), and/or a beta-adrenergic agonist, isoproterenol (ISO). Furthermore, compared to ascorbate-treated controls, TP and ISO increased the synthesis of total cellular protein and PBP by the prostatic renal implants. TP and/or ISO also remodelled the luminal epithelial structure and elevated secretory functions. ISO alone had no effect, however, in castrated animals, indicating that androgen plays a dominant role in the restoration of tissue PBP content. Concomitant to increased PBP content and remodelling of prostatic histomorphology, androgen was also found to raise the depressed levels of beta 2-adrenergic and androgen receptors in the prostatic isografts maintained in intact hosts. In contrast, although an established rat prostatic epithelial cell line (NbE-1) contains high affinity androgen receptor, androgen failed to restore beta-adrenergic receptor as well as PBP content in this cultured cell line. These results, taken together, suggest that a tight coupling between androgen receptor and beta 2-adrenergic receptor pathways may be a prerequisite for PBP expression and functional differentiation in the rat ventral prostate gland.

Characterization of two cis-acting DNA elements involved in the androgen regulation of the probasin gene.

The location and sequence of androgen responsive elements (AREs) in the 5'-flanking DNA of the androgen-regulated rat probasin (PB) gene were determined. The DNA- and steroid-binding domains of the rat androgen receptor [glutathione-S-transferase (GST)-AR1] and the DNA-binding domain and hinge region alone (GST-AR2) were expressed in Escherichia coli as isopropyl-B-D-thioglactopyranoside-induced fusion proteins with GST and purified using glutathione affinity chromatography. Band shift assays indicated that the AR1 peptide was at least five times more effective than AR2 in binding to PB 5'-flanking DNA (-426 to +28), although both gave qualitatively similar patterns and were displaced by anti-AR antibodies. DNase I footprinting experiments revealed two putative AREs: one between positions -236 and -223 (ARE-1) and the other between -140 and -117 (ARE-2). Hormonal regulation of PB was determined by cotransfecting reporter constructions containing the PB 5'-flanking region (-426 to +28) linked to the bacterial chloramphenicol acetyl transferase (CAT) gene with androgen, glucocorticoid, or progesterone receptor expression vectors into human prostatic carcinoma cells (PC-3). PB-CAT gene expression was more effectively induced by androgens than by glucocorticoids or progestins. Both 5'- and 3'-deletion mapping of the PB 5'-flanking DNA revealed that ARE-1 and ARE-2 were required for androgen regulation. A single base mutation in either ARE resulted in a more than 95% loss of androgen induction of CAT. In comparable transfection experiments, the PB hormone-responsive elements showed a greater induction by androgens than did mouse mammary tumor virus or tyrosine aminotransferase elements. Thus, the preferential androgen regulation of the PB gene involves the participation of two different cis-acting DNA elements that bind AR.

Meiotic arrest and germ cell apoptosis in androgen-binding protein transgenic mice.

The fundamental role of androgen-binding protein (ABP) in spermatogenesis remains obscure after nearly 25 yr since its first characterization. In the present investigation, we used a transgenic mouse model that overexpresses rat ABP to examine the potential involvement of this protein in the regulation of processes occurring during spermatogenesis. Specifically, homozygous or heterozygous transgenic mice were analyzed in terms of spermatogenic progression, DNA fragmentation pattern, and germinal cell ploidy status. All animals homozygous for transgenic ABP exhibited an increased accumulation of primary spermatocytes and cells at metaphase with abnormal morphology and localization within the seminiferous epithelium. Analysis of DNA fragmentation by in situ techniques and agarose gel electrophoresis provided evidence for an increased occurrence of apoptosis in the transgenic animals, principally involving pachytene spermatocytes and cells at metaphase. Flow cytometric analysis of the DNA content of isolated germ cells revealed a reduction in the number of haploid cells, an increase in the number of tetraploid cells, and the appearance of a hypotetraploid cell population, consistent with degenerating primary spermatocytes. In mice heterozygous for the transgene, the effects were less prominent, and the degree to which spermatogenesis was compromised correlated with the levels of ABP messenger RNA in individual animals. The present results are interpreted to suggest that ABP can act as a modulator of spermatogenesis by regulating completion of the first meiotic division of primary spermatocytes.

In vitro effects of germ cells on the secretory activity of Sertoli cells recovered from rats of different ages.

Indirect (hypotonically treated culture) and direct (coculture) approaches were used to study the influence of germ cells on androgen-binding protein (ABP) and 17 beta-estradiol secretion by Sertoli cells prepared from 10-, 15-, 20-, and 45-day-old rats. Using these approaches, the mechanisms that govern Sertoli cell-germ cell membrane recognition were shown to be unaltered by the hypotonic treatment, to vary with the age of the Sertoli cell donors, and to be FSH/(Bu)2cAMP dependent in the younger animals (10-20 days old). Hypotonic treatment had no effect on estradiol levels at all ages studied, but resulted in a marked fall of ABP production by Sertoli cells from 20 days onward. However, the relative stimulation of ABP induced by FSH/(Bu)2cAMP (ABP levels in FSH/(Bu)2cAMP/hypotonically treated Sertoli cell cultures vs. ABP in hypotonically treated cultures) was markedly increased at 20 and 45 days, indicating that germ cells influence Sertoli cell responsiveness to FSH/(Bu)2cAMP. The addition of crude germ cell preparation to the Sertoli cell cultures stimulated ABP and inhibited estradiol levels at all ages studied. Moreover, the addition of germ cells to hypotonically treated cultures induced a decrease in the relative ABP response to FSH and (Bu)2cAMP, thus confirming (see above) that germ cells may be involved in the mechanism of Sertoli cell refractoriness to FSH that occurs with advancing age in the rat. Germ cell-stimulated ABP production was higher in adult Sertoli cells than in immature cells. That Sertoli cell responsiveness to germ cells varies with age is further supported by the data showing that whereas spermatocytes stimulated ABP and inhibited estradiol productions at all ages studied, early spermatids only influenced these parameters from 20 days onward. It is concluded that germ cell control over Sertoli cell function in vitro may be of important physiological significance during Sertoli cell maturation in mammals.

Isolation and characterization of rat fetal Sertoli cells.

Sertoli cells were prepared from fetal rats, aged 15, 18, and 21 days. Cultures of these cells can be prepared at a purity of 85% by a method that is widely used to prepare the same cells from postnatal rats. The fetal cells are identical in appearance to postnatal Sertoli cells. Fetal Sertoli cells round up in response to (Bu)2cAMP, but not in response to FSH. Protein synthesis in the cells is not stimulated by (Bu)2cAMP or FSH. However, incorporation of [35S]methionine into secreted proteins appearing in the incubation medium is stimulated by the cyclic nucleotide, but not by FSH. Reproducible patterns for the incorporation of [35S]methionine into cellular and secreted proteins are presented as autoradiograms of one- and two-dimensional electrophoretograms. These autoradiograms show that the response of secreted proteins to (Bu)2cAMP is a general one; most or all proteins participate in the response. No clear differences were observed in the nature of the proteins synthesized when cells from fetal rats of 15, 18, and 21 days of age were compared. Fetal Sertoli cells produced approximately 2 nmol lactate/10(6) cells.h, which is approximately one fifth of the amount produced by postnatal cells. Lactate production was increased by the addition of FSH or (Bu)2cAMP to the medium. Fetal Sertoli cells also synthesize androgen-binding protein, and this activity is not increased by either FSH or (Bu)2cAMP.

Regulation of Sertoli cell function and differentiation through the actions of a testicular paracrine factor P-Mod-S.

The current study was designed to investigate the actions of the testicular paracrine factor P-Mod-S on Sertoli cell function and differentiation. Transferrin production by Sertoli cells was stimulated by P-Mod-S to a greater extent than any individual regulatory agent and in a manner similar to a combination of FSH, insulin, retinol, and testosterone (FIRT). P-Mod-S had an additive response in combination with FIRT. The increase in transferrin production with a combination of P-Mod-S and FIRT is the highest level of stimulation (up to 8-fold) observed. These profound effects of P-Mod-S on Sertoli cell function implied a potential unique mechanism of action for the paracrine factor. FSH and FIRT significantly stimulated cAMP levels with both 60-min and 72-h treatments. In contrast, P-Mod-S had no effect on cAMP levels with a 60-min treatment and only a small increase with a 72-h treatment. Interestingly, P-Mod-S stimulated cGMP levels that remained above basal levels up to 72 h of treatment. FSH had no effect on cGMP levels. P-Mod-S did not affect inositol phosphate hydrolysis with treatments between 15 and 60 min. The actions of P-Mod-S on cGMP levels influenced Sertoli cell function on a molecular level. Northern blot analysis indicated that P-Mod-S and FIRT both stimulated the apparent levels of the 2.6-kilobase transcript of transferrin and the 1.7-kilobase transcript of androgen-binding protein. A solution hybridization procedure was used to quantitate the influence of P-Mod-S on Sertoli cell gene expression. P-Mod-S stimulated steady state levels of both transferrin and androgen-binding protein message approximately 2-fold, similar to the effects of FIRT. Both forms of P-Mod-S had similar biological activities and mechanisms of action. P-Mod-S (A) and P-Mod-S (B) both stimulated cGMP, altered Sertoli cell gene expression, and had profound effects on transferrin production. Although slightly different biochemically, both forms of P-Mod-S appear to be functionally similar. Combined observations indicate that the paracrine factor produced by peritubular cells, P-Mod-S, acts on Sertoli cells in part through a cGMP-mediated response to influence the expression of specific genes which subsequently have profound effects on Sertoli cell function and differentiation.

Hormonal regulation of androgen-binding protein in the rat.

Effects of gonadotropins and gonadal steroids on androgen-binding protein (ABP) production by the testis and its secretion into the blood and transport into the epididymis were studied in 20- and 30-day-old rats. These animals had been treated with hCG, FSH, the Nal-Glu GnRH antagonist [Ac-D2Nal1,D4ClDPhe2,D3Pal3,Arg5,DGlu6(AA) ,DAla10]LHRH (antagonist), testosterone propionate, or estradiol benzoate, alone or in combination, for 10 days before assessment of ABP. Antagonist administration suppressed the testicular content (nanograms per organ) of ABP to below control (untreated) levels in both age groups. When hCG or testosterone was given along with the antagonist, they overcame the effect of the antagonist, and the resultant ABP values exceeded untreated control levels in both the 20- and 30-day-old rats. Treatment of rats with these hormones in the absence of the GnRH antagonist also elevated the ABP content of the testis above that of untreated controls. FSH administered with antagonist was able to prevent the antagonist-induced suppression of testicular ABP content. When rats were treated with FSH alone, the content of ABP in the testis was increased above untreated control levels in the 30-day-old group, but not in the 20-day-old group. The simultaneous administration of FSH and hCG did not result in an increase in testicular ABP content above that caused by hCG or testosterone alone. The increase in the ABP content of the testis caused by FSH administration was only about one sixth that caused by hCG or testosterone. Since testosterone or hCG, even in the presence of antagonist, was able to maximally stimulate ABP production by the testis of both age groups, we conclude that testosterone is the major in vivo regulator of its synthesis. Only combined treatment with hCG and FSH was able to increase transport of ABP into the epididymis of 20-day-old rats. All treatments that increased the testicular content of ABP in the 30-day-old rats also increased its transport into the epididymis. Treatments that drastically reduced the content of ABP in the testis of 20-day-old rats (antagonist, estradiol, estradiol plus antagonist) also reduced ABP secretion into the serum. Only treatment with estradiol reduced the secretion of ABP into the serum of 30-day-old rats. None of the treatments increased the ABP secretion into the bloodstream above untreated control levels.

Androgens transcriptionally regulate the expression of cystatin-related protein and the C3 component of prostatic binding protein in rat ventral prostate and lacrimal gland.

In this report, it is demonstrated that the C3 component of prostatic binding protein (PBP) is also expressed and androgen regulated in the exorbital lacrimal gland, as shown previously for cystatin-related protein (CRP), another abundant secretory protein from the ventral prostate. The presence of C3 messenger RNA (mRNA) could be demonstrated by both Northern blot hybridization and PCR amplification and sequencing. The mRNAs encoding the C1 and C2 components of PBP, however, were undetectable. At the protein level, the C3 component in the lacrimal gland is glycosylated and linked by disulfide bridges to a new 10-kDa component not reacting with the PBP antiserum. As shown previously for CRP, the expression of C3 in the lacrimal gland requires the simultaneous presence of androgens and a functional androgen receptor. The effects of castration and androgen treatment on CRP and C3 mRNA concentrations were studied by Northern blot and dot blot hybridization; effects on transcription rates were determined by nuclear run-on assay. Two days after castration, the relative abundance of CRP mRNA had declined significantly (P < 0.01) to 10.5 +/- 1.5% (+/-SEM) of precastration levels in the prostate and to 14.5 +/- 8.0% in the lacrimal gland; the transcription rates declined to 14.3% and 10.0%, respectively. The C3 mRNA level and transcription rate in the prostate showed a more moderate decrease (P < 0.05) to 40.6 +/- 8.5% and 41.7%, but were hardly measurable in the lacrimal gland. Androgen administration resulted in a rapid increase in the transcription rates, which reached or exceeded control levels after 6-9 h of treatment and clearly preceded the increase in mRNA levels. It is concluded that the lacrimal gland, which can be studied conveniently in female and long term androgen-depleted animals offers a suitable model for the study of androgen-regulated gene expression.

Regulation of Sertoli cell differentiated function: testicular transferrin and androgen-binding protein expression.

The regulation of Sertoli cell function was investigated through an examination of the effects of various hormones, regulatory agents, and culture conditions on testicular transferrin and androgen-binding protein (ABP) synthesis and steady state levels of mRNA. FSH stimulated both transferrin and ABP production 2-fold above control levels. Interestingly, FSH had a differential effect on transferrin and ABP mRNA levels, with 1.25- and 2.0-fold respective increases in steady state levels of mRNA. Insulin and retinol stimulated both transferrin and ABP synthesis in a similar manner. Testosterone had no significant effect on either transferrin or ABP mRNA levels or synthesis. Maximum stimulation of both transferrin and ABP production occurred when Sertoli cell cultures were treated with a combination of FSH, insulin, and retinol, which resulted in a greater than 4-fold stimulation of synthesis and 2-fold stimulation of gene expression. Optimal transferrin and ABP secretion occurred between days 4-6 of Sertoli cell culture and subsequently declined. Sertoli cell number decreased with time in culture, such that approximately a 50% loss of cells was observed after 10 days of culture. The responsiveness of Sertoli cells to regulatory agents was altered by cell density, with a maximum responsiveness achieved at a density of 12 micrograms DNA/2 cm2 for both transferrin and ABP. As the cell density deviated from this level the responsiveness of cells to regulatory agents decreased and approached control values. These observations indicate that the culture conditions and the method of data normalization are important parameters in an analysis of the hormonal regulation of Sertoli cell function. FSH actions on Sertoli cells increased both cellular and excreted cAMP levels but had no effect on cGMP levels. (Bu)2 cAMP affected transferrin and ABP mRNA levels and synthesis in a similar manner, with approximately a 3-fold increase in synthesis and a 1.5-fold increase in steady state levels of mRNA. The minimum and maximum effective concentrations of (Bu)2AMP for both proteins were 1 and 10 microM, respectively. Observations imply that regulatory agents that act via a cAMP-mediated signal transduction mechanism, such as FSH, will probably have similar actions on transferrin and ABP production. In addition, data obtained with insulin and retinol indicate that transferrin and ABP production can be similarly regulated with cAMP-independent signal transduction mechanisms. Results indicate that transferrin and ABP mRNA levels and synthesis are regulated in a coordinate manner with the regulatory agents and culture conditions evaluated.(ABSTRACT TRUNCATED AT 400 WORDS)

Heregulins or neu differentiation factors and the interactions between peritubular myoid cells and Sertoli cells.

Interactions between (mesenchymal) peritubular myoid cells and (epithelial) Sertoli cells play an important role in the control of Sertoli cell function and spermatogenesis. The factors involved, however, have only partially been identified. Heregulins or neu differentiation factors (NDFs) mediate mesenchymal-epithelial interactions in a variety of tissues, but their role in the testis has not been investigated. Here we demonstrate that recombinant human heregulin-alpha (Her-alpha) and Her-beta stimulate transferrin and androgen-binding protein production by cultured rat Sertoli cells up to 2.5-fold. These effects are more pronounced than those of previously identified growth factors active in this assay, such as insulin-like growth factor I and basic fibroblast growth factor. Combination with these factors results in additive effects and in marked morphological changes in the Sertoli cell cultures, including formation of tubule-like structures. Stimulation of androgen-binding protein secretion is paralleled by increased levels of the corresponding messenger RNA. This parallelism was less consistent for transferrin. Semiquantitative RT-PCR indicates that the expression of NDF-alpha and NDF-beta is more pronounced in peritubular cells than in Leydig or Sertoli cells. Conversely, the main receptors for heregulins/NDFs, HER-3 and HER-4, are predominantly expressed in Sertoli cells. A displacement assay confirms the presence of high-affinity binding sites for [125I]Her-beta on intact Sertoli cells and reveals parallel displacement curves for Her-beta, Her-alpha, and concentrated peritubular cell-conditioned medium (PTCM; estimated ED50 values: 1 ng/ml, 50 ng/ml, and 130 microg protein/ml, respectively), indicating that peritubular cells secrete one or more factors able to compete for heregulin receptors. It is concluded that heregulins/NDFs may play a role in mesenchymal-epithelial interactions in the testis. Estimates of the concentrations of heregulins in PTCM, however, make it unlikely that they contribute significantly to the effects observed with low concentrations of PTCM and ascribed to the putative mediator PModS (peritubular factor that modulates Sertoli cell function). Further investigations will be required to define the exact role of heregulins in the testis.

Androgenic induction of cystatin-related protein and the C3 component of prostatic binding protein in primary cultures from the rat lacrimal gland.

Cystatin-related protein (CRP) and the C3 component of prostatic binding protein (C3) are synthesized in vivo under androgen control in the lacrimal gland and ventral prostate of adult male rats [1,2]. Androgen administration to female or 7-day castrated male rats, which do not express CRP, can induce its synthesis [3]. In this study, we show androgen-dependent expression of CRP1 and C3 in primary cultures of acinar cells of the lacrimal gland of female rats. Addition of androgens to the culture medium results in the synthesis and secretion of both proteins in a time- and dose-dependent way. Estradiol or progesterone are unable to induce their expression. Dexamethasone in low concentrations and present as a basal component of the serum free defined medium, is needed to sensitize the culture system for androgens. In high concentrations, this synthetic glucocorticoid seems to play a similar role as androgens in CRP1 and C3 regulation.