Identification, characterization, and evolution of a primate beta-defensin gene cluster.

Defensins are members of a large diverse family of cationic antimicrobial peptides that share a signature pattern consisting of six conserved cysteine residues. Defensins have a wide variety of functions and their disruption has been implicated in various human diseases. Here we report the characterization of DEFB119-DEFB123, five genes in the human beta-defensin cluster locus on chromosome 20q11.1. The genomic structures of DEFB121 and DEFB122 were determined in silico. Sequences of the five macaque orthologs were obtained and expression patterns of the genes were analyzed in humans and macaque by semiquantitative reverse transcription polymerase chain reaction. Expression was restricted to the male reproductive tract. The genes in this cluster are differentially regulated by androgens. Evolutionary analyses suggest that this cluster originated by a series of duplication events and by positive selection. The evolutionary forces driving the proliferation and diversification of these defensins may be related to reproductive specialization and/or the host-parasite coevolutionary process.

Primate epididymis-specific proteins: characterization of ESC42, a novel protein containing a trefoil-like motif in monkey and human.

Epididymal secreted proteins promote sperm maturation and fertilizing capacity by interacting with sperm during passage through the epididymis. Here we investigate the molecular basis of sperm maturation by isolating cDNA clones for novel epididymis-specific expressed sequences. Thirty-six novel cDNAs were isolated and sequenced from a subtracted Macaca mulatta epididymis library. The clones encode proteins with a range of motifs characteristic of protein-modifying enzymes, protease inhibitors, hydrophobic ligand-binding and transport proteins, extracellular matrix-interacting proteins, and transcription regulatory factors. The full length coding sequences were obtained for 11 clones representing a range of abundance levels. Expression of each is regionally localized and androgen regulated. The most abundant, ESC42, contains a cysteine-rich region similar to the signature binding domain of the trefoil family of motogenic wound repair proteins. The monkey and human proteins are nearly 90% identical. Immunohistochemical staining revealed that the protein is most abundant in the epithelium of the caput and is also present in the lumen and bound to sperm. The ESC42 gene, located on chromosome 20q11, contains two exons encoding two nearly identical predicted signal peptides and a third exon encoding the rest of the protein.

Cloning and sequencing of human Eppin: a novel family of protease inhibitors expressed in the epididymis and testis.

In this report we describe the discovery of Eppin (Epididymal protease inhibitor), a gene on human chromosome 20 expressing three mRNAs encoding two isoforms of a cystine-rich protein containing both Kunitz-type and WAP-type four disuffide core protease inhibitor consensus sequences. Analysis of Eppin's genomic sequence from chromosome 20q12-13.2 predicts the existence of all three splice variants of Eppin and that all the exons conform to the AG/GT splicing rule. The presence of single bands on a Southern blot of human genomic DNA suggests that Eppin is a single copy gene. TATA box transcription initiation sites are present for both of the different Eppin 5' UTRs and examination of the promoter region 1800 bp upstream of the start codon revealed a number of putative transcription enhancer binding sites typical of genes expressed in the epididymis or testis. Northern blot and tissue specific PCR data indicate Eppin-1 is expressed only in the testis and epididymis; Eppin-2 is expressed only in the epididymis and Eppin-3 only in the testis. Antiserum prepared against recombinant EPPIN recognizes several strong bands on Western blots of human epididymal extracts from the caput and corpus regions. Immunohistochemistry indicates a strong pattern of expression by the ciliated cells of the efferent ducts and strong staining of ejaculated spermatozoa. Eppin represents the first member of a family of protease inhibitors characterized by dual inhibitor consensus sequences, both WAP-type and Kunitz-type consensus sequences. A second family member is predicted to exist on chromosome 20 approximately 4 kb downstream from Eppin's exon I, which has two WAP-type sequences and one Kumtz-type consensus sequence.

Comparison of four automated hematology analyzers.

OBJECTIVE: To compare four automated hematology analyzers for efficiency and sensitivity. DESIGN: Four automated hematology analyzers were compared in a side by side study: Bayer ADVIA 120 (Bayer Diagnostic Division, Tarrytown, NY), Beckman Coulter GEN S (Beckman Coulter, Brea, CA), Abbott CELL DYN 3500 and CELL DYN 4000 (Abbott Diagnostics, Santa Clara, CA). 164 specimens were analyzed for cell counts, indices, and the automated WBC differential (DLC). Tallies were kept of all interventions, defined as any parameter necessitating examination of a stained blood smear by a clinical laboratory scientist. A 400-cell manual differential was performed on each specimen and used as the reference to prepare truth tables for each type of WBC. PATIENTS: Specimens comprised regular runs from this tertiary care teaching hospital. These included inpatients, outpatients, and oncology patients, including bone marrow transplant patients. MAIN OUTCOME MEASURES: Results from the truth tables were used for calculating sensitivity and efficiency for each analyzer. Each DLC parameter was analyzed for variance using the one-way ANOVA test. RESULTS: No intervention was required for 103 of 164 specimens for the CELL DYN 3500; the ADVIA gave 70 reportable DLCs without intervention, the GEN S provided 91 and the CELL DYN 4000 resulted in 117 of 164 DLCs without intervention. Agreement or efficiency was 65% for the CELL DYN 3500, 41% for the ADVLA, 58% for the GEN S, and 79% for the CELL DYN 4000. Sensitivity was 67% for the CELL DYN 3500, 86% for the ADVIA, 76% for the GEN S, and 71% for the CELL DYN 4000. Probability of significant variation was as follows for each parameter: % neutrophil 0.8747, % lymphocyte 0.8830, % monocyte 0.0296, % eosinophil 0.7903, and % basophil <.0001. CONCLUSION: The analyzers tested were acceptable for routine laboratory work. Selection would depend on individual need with respect to sensitivity and efficiency. The clinical significance of disagreement between the DLC and the manual differential remains to be determined.

Thyroid receptor activator molecule, TRAM-1, is an androgen receptor coactivator.

An androgen receptor (AR) interacting protein was isolated from a HeLa cell complementary DNA library by two-hybrid screening in yeast using the AR DNA and ligand binding domains [amino acids (aa) 481-919] as bait. AR binding of the protein in yeast was dependent on the presence of testosterone or dihydrotestosterone (DHT). The isolated protein is identical to thyroid receptor activator molecule TRAM-1 but lacking aa 1-458. TRAM-1 is a steroid receptor coactivator-3 (SRC-3) subtype. In affinity matrix assays, 35S-labeled TRAM-1 bound the GST-AR ligand binding domain (aa 624-919) and GST-AR N-terminal and DNA binding domains (aa 1-660), but not the GST-AR DNA binding domain (aa 544-634) alone. Coexpression of TRAM-1 increased DHT-dependent AR transactivation 5-fold and constitutive activity of AR (aa 1-660) N-terminal and DNA-binding domains increased 9-fold. Full-length TRAM-1 (aa 1-1424) and the partial (aa 459-1424) were AR and GR coactivators as was SRC-1. In human testis, immunostaining of SRC-3 colocalized with AR in nuclei of Sertoli cells and peritubular myoid cells, indicating it could function as an AR coactivator in these cells. SRC-3 was also present in nuclei of spermatogenic cells where AR was not expressed, suggesting it might also be a coactivator with other nuclear receptors that regulate spermatogenesis.

HE2beta and HE2gamma, new members of an epididymis-specific family of androgen-regulated proteins in the human.

HE2 is an epididymis-specific sperm-binding secretory protein. We isolated a family of HE2-related complementary DNAs from a human caput/corpus library. The transcripts code for identical 71-amino acid N-termini and different C-termini, and 5'- and 3'-untranslated regions. Compared with the original HE2, HE2beta and HE2gamma proteins have a 25-amino acid deletion near the C-terminus, and HE2gamma isoforms have a second deletion. These frame-shifting deletions result in C-termini differing in length, amino acid sequence, including number of cysteines, and isoelectric point. Identical sequences and deletion start and stop points indicate the HE2 isoforms are derived from alternative splicing of 8 or more exons of a single gene. Northern hybridization revealed that the 0.9-kb messenger RNA (mRNA) is most abundant in human caput; there is much less of it (20%) in corpus and little (<5%) in cauda. In castrated Macaca mulatta, HE2 mRNA decreased to 10% of sham-operated levels. Testosterone replacement maintained HE2 mRNA 3- to 5-fold higher than castrate levels, indicating its androgen dependence. Immunohistochemical staining revealed that the beta1 form is highly expressed in principal cells of the initial segment and caput. It is secreted into the lumen and binds to the sperm surface in the postacrosomal and neck regions. The beta2 form is expressed in principal cells primarily in efferent ducts.

Protein inhibitor of activated STAT-1 (signal transducer and activator of transcription-1) is a nuclear receptor coregulator expressed in human testis.

An androgen receptor (AR) interacting protein was isolated from a HeLa cell cDNA library by two-hybrid screening in yeast using the AR DNA+ligand binding domains as bait. The protein has sequence identity with human protein inhibitor of activated signal transducer and activator of transcription (PIAS1) and human Gu RNA helicase II binding protein (GBP). Binding of PIAS1 to human AR DNA+ligand binding domains was androgen dependent in the yeast liquid beta-galactosidase assay. Activation of binding by dihydrotestosterone was greater than testosterone > estradiol > progesterone. PIAS1 binding to full-length human AR in a reversed yeast two hybrid system was also androgen dependent. [35S] PIAS1 bound a glutathione S-transferase-AR-DNA binding domain (amino acids 544-634) fusion protein in affinity matrix assays. In transient cotransfection assays using CV1 cells with full-length human AR and a mouse mammary tumor virus luciferase reporter vector, there was an androgen-dependent 3- to 5-fold greater increase in luciferase activity with PIAS1 over that obtained with an equal amount of control antisense cDNA or mutant PIAS1. Constitutive transcriptional activity of the AR N-terminal+DNA binding domain was increased 6-fold by PIAS1. PIAS1 also enhanced glucocorticoid receptor transactivation in response to dexamethasone but inhibited progesterone-induced progesterone receptor transactivation in the same assay system. mRNA for PIAS1 was highly expressed in testis of human, monkey, rat, and mouse. In rat testis the onset of PIAS1 mRNA expression coincided with the initiation of spermatogenesis between 25-30 days of age. Immunostaining of human and mouse testis with PIAS1-specific antiserum demonstrated coexpression of PIAS1 with AR in Sertoli cells and Leydig cells. In addition, PIAS1 was expressed in spermatogenic cells. The results suggest that PIAS1 functions in testis as a nuclear receptor transcriptional coregulator and may have a role in AR initiation and maintenance of spermatogenesis.

Androgen receptor expression in androgen-independent prostate cancer is associated with increased expression of androgen-regulated genes.

The human prostate cancer (CaP) xenograft, CWR22, mimics human CaP. CWR22 grows in testosterone-stimulated nude mice, regresses after castration, and recurs after 5-6 months in the absence of testicular androgen. Like human CaP that recurs during androgen deprivation therapy, the recurrent CWR22 expresses high levels of androgen receptor (AR). Immunohistochemical, Western, and Northern blot analyses demonstrated that AR expression in the androgen-independent CWR22 is similar to AR expression in the androgen-dependent CWR22 prior to castration. Expression of prostate-specific antigen and human kallikrein-2 mRNAs, two well-characterized androgen-regulated genes in human CaP, was androgen dependent in CWR22. Despite the absence of testicular androgen, prostate-specific antigen and human kallikrein-2 mRNA levels in recurrent CWR22 were higher than the levels in regressing CWR22 tumors from 12-day castrate mice and similar to those in the androgen-stimulated CWR22. Other AR-regulated genes followed a similar pattern of expression. Differential expression screening identified androgen regulation of alpha-enolase and alpha-tubulin as well as other unknown mRNAs. Insulin-like growth factor binding protein-5, the homeobox gene Nkx 3.1, the AR coactivator ARA-70, and cell cycle genes Cdk1 and Cdk2 were androgen regulated in CWR22. In recurrent CWR22, the steady-state levels of all these AR-dependent mRNAs were similar to those in the androgen-stimulated CWR22, despite the absence of testicular androgen. Expression of AR and AR-regulated genes in the androgen-deprived recurrent CWR22 at levels similar to the androgen-stimulated CWR22 suggests that AR is transcriptionally active in recurrent CWR22. Induction of these AR-regulated genes may enhance cellular proliferation in relative androgen absence but through an AR-dependent mechanism. Alternatively, in androgen-independent tumors, induced expression of the AR-regulated gene network might result from a non-AR transcription control mechanism common to these genes.

Androgen and glucocorticoid receptors in the stroma and epithelium of prostatic hyperplasia and carcinoma.

Differences in stromal and epithelial cell staining for androgen and glucocorticoid receptors (ARs and GRs) were investigated in 20 patients with clinically localized prostatic carcinoma treated by radical prostatectomy. Sections of benign prostatic hyperplasia and prostatic carcinoma from each patient were stained with antibodies to AR and GR using an avidin-biotin peroxidase technique. The specificity of the GR immunoreactivity was established in benign prostatic hyperplasia and prostatic carcinoma by immunohistochemistry using the GR antibody absorbed with synthetic peptide and Western blotting. Nuclear staining intensity and percentage of nuclei stained were summed to obtain AR and GR immunostaining scores. AR staining of prostatic carcinoma epithelial [103 +/- 58 (SD)] and stromal (126 +/- 48) nuclei was less than in benign prostatic hyperplasia (142 +/- 47 and 169 +/- 56; paired Student's t tests, P = 0.02 and P = 0.01); however, no difference in staining intensity occurred between stroma and epithelium in either tissue type. GR stained intensely in stromal cells from benign prostatic hyperplasia (189 +/- 50) and prostatic carcinoma (163 +/- 60). However, prostatic carcinoma epithelial cells (34 +/- 57) had low levels of glucocorticoid receptor staining (P < 10(-7)), and benign prostatic hyperplasia epithelium (74 +/- 51) was intermediate. In most patients, GR could not be detected in nuclei of prostatic carcinoma epithelial cells but was undiminished in stromal cell nuclei. There was no relationship by multivariate regression analysis between AR or GR staining and age, serum prostate-specific antigen, Gleason grade, or pathological stage. In comparison with AR, the greater variability of GR staining in epithelium versus stroma of prostatic carcinoma warrants further study of GR, particularly in the area of stromal-epithelial interaction.

Stage-specific expression of B cell translocation gene 1 in rat testis.

B Cell translocation gene 1 (BTG1) is a member of a new family of putative antiproliferative factors. They are characterized by their rapid, but transient, expression in response to factors that induce growth arrest and subsequent differentiation. In immature rat Sertoli cell cultures, BTG1 messenger RNA (mRNA) increases rapidly after FSH stimulation. We obtained the full-length coding sequence of rat BTG1 complementary DNA for Northern blot analysis and in situ hybridization to determine the temporal expression and spatial distribution of BTG1 mRNA in the rat testis. Northern analysis of isolated adult germ cells and in situ hybridization analysis of adult seminiferous epithelium demonstrated that BTG1 expression was first evident in late primary spermatocytes. The level of BTG1 mRNA was also elevated in secondary spermatocytes, but was maximal in postmeiotic round spermatids where levels were 5 times the background. BTG1 mRNA was not detectable in cells in the M phase of meiosis or spermatids undergoing nuclear elongation and condensation. The oscillation of BTG1 expression from the late prophase of the first meiotic division through spermatozoa release suggests BTG1 involvement in spermatogenesis. High levels of BTG1 mRNA at entry into terminal spermatid differentiation suggests a role consistent with that proposed for the BTG1 family of antiproliferative factors.

Follicle-stimulating hormone regulation of AP-1: inhibition of c-jun and stimulation of jun-B gene transcription in the rat Sertoli cell.

The proto-oncogenes c-fos and c-jun and the related jun-B encode the components of transcription factor, AP-1, a heterodimeric DNA-binding protein that mediates hormone and growth factor-regulated gene expression. In the rat Sertoli cell, FSH rapidly inhibited c-jun gene expression while it stimulated c-fos and jun-B as well as the expression of the more slowly responding, tissue plasminogen activator (tPA) and inhibin alpha-subunit. These early effects of FSH were not inhibited by cycloheximide. Nuclear run-off analyses demonstrated that the FSH-dependent decline in c-jun and increases in c-fos, jun-B, tPA and inhibin alpha-subunit mRNAs were regulated at the transcriptional level. The rates of degradation of c-fos, c-jun and jun-B mRNAs were unaffected by FSH while tPA and inhibin alpha-subunit mRNAs were stabilized. After 8 h of FSH treatment, the transcription of all five genes returned to basal rates. These data demonstrate immediate-early regulation by FSH of the expression of genes encoding components of the transcription factor, AP-1.

Regulation of c-fos, c-jun and jun-B messenger ribonucleic acids by angiotensin-II and corticotropin in ovine and bovine adrenocortical cells.

Previous work has shown that corticotropin (ACTH) and angiotensin-II (A-II), in addition to their acute steroidogenic effects, exert long-term influences on adrenal cell differentiated function, stimulatory or inhibitory, respectively. Certain nuclear proto-oncogenes have been implicated in the regulation of gene expression in many cell systems. We have investigated the effects of ACTH and A-II on the levels of c-fos, c-jun, and jun-B messenger RNAs (mRNAs), in bovine and ovine (OAC) adrenal fasciculata cells. In both cell types ACTH produced time- (maximum at 1 h) and dose-dependent (ED50 congruent to 10(-12) M) increase in c-fos (2- to 4-fold) and jun-B (10- to 20-fold) mRNA levels but did not affect c-jun. The concentrations required to induce half-maximal mRNA accumulation and cortisol production were similar. A-II also produced a dose-dependent increase in c-fos and jun-B mRNAs but also in c-jun in both cell types, despite the fact that OAC are resistant to the steroidogenic action of the hormone. The stimulatory effects of A-II on c-fos mRNA were higher than those produced by ACTH, whereas the effects on jun-B were similar but ACTH abolished (OAC) or decreased (bovine adrenal fasciculata cells) the stimulatory effects of A-II on c-jun mRNA. The effects of ACTH and A-II on cortisol production and proto-oncogene mRNAs were in part mimicked by 8 Bromo-cAMP and the phorbol ester phorbol-12-myristate-13 acetate plus calcium ionophore A23187, respectively. In the presence of cycloheximide, which blocks the steroidogenic effects of both hormones, proto-oncogene mRNAs were superinduced by both hormones. This result, together with the fact that dexamethasone failed to affect the mRNA levels suggests that the stimulatory effects of ACTH and A-II on proto-oncogene expression were not related to an autocrine/intracrine action of cortisol. Taken together, these findings show that the proto-oncogene mRNAs in normal adrenal cells are regulated by ACTH and A-II, acting through different intracellular pathways. They also demonstrate differential responsiveness of the Jun family to both hormones. Thus, the opposite long-term action of ACTH and A-II on adrenal cell differentiated function could be mediated by its different initial effects on proto-oncogene expression, in particular in the members of the Jun family.

Regulation of c-fos, c-jun, jun-B, and c-myc messenger ribonucleic acids by gonadotropin and growth factors in cultured pig Leydig cell.

The nuclear protooncogenes have been implicated in the coordinate regulation of gene expression during cell proliferation and differentiation. Previous work has shown that LH and human h CG as well as several growth factors including epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), transforming growth factor-beta, and insulin-like growth factor-I play a role in Leydig cell differentiated functions. To evaluate the possibility that protooncogenes mediate long term effects of these factors, their action on the levels of c-fos, c-jun, jun-B, and c-myc messenger (m) RNAs was studied. hCG (10(-9) M) produced a time-dependent increase in c-fos (9-fold), jun-B (18-fold) and c-myc (5-fold) mRNA levels but did not affect c-jun. The concentration of hCG required for half-maximal stimulation (ED50 = 7 +/- 4 x 10(-12) M) was similar to that required to induce half-maximal testosterone production. At optimal concentrations, the effects of EGF and bFGF on c-fos and jun-B mRNAs were lower than those induced by hCG, but their effects on c-myc mRNA were higher. In addition, they stimulated c-jun. Moreover, EGF and bFGF potentiated the effects of hCG on c-fos and jun-B, whereas hCG potentiated the action of growth factors on c-jun. Transforming growth factor-beta increased only jun-B mRNA, whereas insulin-like growth factor I increased c-fos, jun-B, and c-myc but less effectively than hCG. Lastly, the phorbol ester phorbol 12-myristate 13-acetate increased the level of the four protooncogene mRNAs, and its effects on c-fos and c-myc were significantly higher than those produced by hCG. These data indicate that the regulation of protooncogene mRNAs in normal Leydig cells is multifactorial. They also show differential responsiveness of the members of the Jun family to several factors. Our results are consistent with the hypothesis that the Fos and Jun families of regulatory proteins could play a role in mediating long term responses to the complex array of hormones and growth factors to which Leydig cells are exposed in vivo.

Presence of osteonectin/SPARC in mandibular condylar cartilage of the rat.

Osteonectin/SPARC was most abundantly present in adherent osseous tissue of the cartilage explants. In cartilage explants with ossification fronts, it appears to be present in hypertrophic chondroblasts and in the mineralised extracellular cartilage matrix. In a number of cartilage explants it could be demonstrated in the fibroblastic cells of the perichondrium and, intra- and extracellularly, in cartilage located adjacent to the perichondrium. In young mandibular condylar cartilage (20 days post-conception up to 7 days of age) osteonectin/SPARC was characteristically present in the transitional zone, a small area of differentiating skeletoblasts. In cartilage, osteonectin/SPARC might play a role in the process of mineralisation and subsequent replacement by bone. It seems to be an important marker of skeletal differentiation processes.

Regulation of c-fos messenger ribonucleic acid by fibroblast growth factor in cultured Sertoli cells.

The data reported here demonstrate that basic FGF and Sertoli-cell-derived FGF have rapid stimulatory effects on c-fos mRNA levels. Basic FGF appears to utilize a signal transduction pathway that is distinct from that used by FSH and serum but similar in its potency and transiency. This is consistent with other reports in the literature on FGFs mechanism of action. For example, a monoclonal antibody to phosphatidyl 4,5-biphosphate will block the effects of PDGF on thymidine incorporation into NIH 3T3 cells but has no effect on basic FGF. Our data support the emerging possibility of a novel pathway mediating FGF actions. A similar precedent has been established for serotonin-induced DNA synthesis in smooth muscle cells. The rat Sertoli cell, with both FSH and FGF rapidly inducing c-fos, is an excellent model system for addressing the mechanism of action of basic FGF, the specificity of the c-fos response and the role of FGF in the reproductive system. Stimulation of c-fos mRNA by basic FGF in the cultured Sertoli cell presents questions regarding the role of FGF and c-fos in the male reproductive system. Basic FGF has been shown to stimulate cell division and plasminogen activator activity in cultured immature porcine Sertoli cells. Plasminogen activator may play a critical role in the tissue remodeling required for spermiogenesis. Interestingly, fos has been shown to be expressed preferentially in pachytene spermatocytes in the mouse. These observations taken together with the finding that basic FGF mRNA levels in Xenopus oocytes are markedly elevated, suggests that basic FGF may be important for Sertoli cell function, spermatogonial cell proliferation and subsequent meiotic and sperm maturational events.

Follicle-stimulating hormone induces transient expression of the protooncogene c-fos in primary Sertoli cell cultures.

The expression of the protooncogene c-fos has been associated with the transduction of cell surface stimuli into changes in nuclear function. To evaluate the possibility that this protooncogene plays a role in the gonadotropin-dependent gene regulation, the effect of FSH on the expression of c-fos was studied in primary Sertoli cell cultures. Sertoli cells were stimulated for different time intervals with FSH and c-fos mRNA levels measured by Northern RNA blot analysis. FSH treatment increased c-fos mRNA transiently with a maximal stimulation reached in 1 h. The level of c-fos mRNA returned to basal level within 4-6 h. The induction of c-fos mRNA was dependent on the concentration of FSH used with an ED50 of 3-5 ng/ml ovine FSH-16. A similar increase in c-fos expression was induced with highly purified hFSH. The c-fos mRNA was also elevated after treatment of the Sertoli cell with (Bu)2cAMP and forskolin. (Bu)2cAMP treatment led to a sustained induction of c-fos mRNA, with increased mRNA levels being maintained after 12 h. The FSH-dependent induction of c-fos mRNA was still present in cells treated for 3 h with cycloheximide, but it was greatly reduced by actinomycin D pretreatment. These data indicate that FSH induces a transient expression of c-fos in cultured Sertoli cells. This induction is probably mediated by cAMP and likely involves an increased transcription of the c-fos gene. Early expression of this gene might be an intermediate step required for gonadotropin-dependent regulation of expression of other genes.

Regulation of ovine pituitary glycoprotein hormone alpha subunit mRNA by 17 beta-estradiol in cell culture.

Messenger RNA (mRNA) activity for the alpha glycoprotein subunit in ovine pituitary cell cultures was quantified using a wheat germ translation assay. [35S] Cysteine alpha translation product normally accounted for 0.3-0.9% of total 35S-product in translations of total culture RNA. Treatment of cultures with 1 nM 17 beta-estradiol (E2) decreased alpha mRNA activity by 50% and 92% after 1.8 and 8 days, respectively. Alpha mRNA activity decreased by 1/2 in 51 h, which is about three times slower than the rate of decrease reported for follicle-stimulating hormone (FSH) beta mRNA activity in similarly treated cultures (Alexander and Miller, 1982). The effective dose (ED50) for E2 in vitro was 30 pM (8 pg/ml), which is within the physiological range for ewes. These results indicate that E2 can act directly on the ovine pituitary to decrease alpha mRNA activity coordinately with mRNA activity for FSH beta.

Regulation of beta-chain mRNA of ovine follicle-stimulating hormone by 17 beta-estradiol.

17 beta-Estradiol (E2) rapidly and reversibly decreases the synthesis of follicle-stimulating hormone (FSH) in primary dispersed cell cultures of ovine pituitaries. Similarly, E2 also causes a decrease in the messenger RNA for the beta subunit of ovine FSH (FSH beta-mRNA) as measured in an in vitro translation assay. These results are consistent with the concept that E2 directly regulates either transcription of the FSH beta gene or processing of FSH beta-mRNA in sheep. Of additional interest is the observation that pituitary cultures from various species respond differently to E2 in terms of FSH synthesis. For example, E2 stimulates FSH synthesis in rat pituitary cultures, has no effect in similar rabbit cultures, and inhibits FSH synthesis in ovine cultures. Thus, a set of eukaryotic 'mutants' may exist to aid studies of the effect of E2 on FSH synthesis and secretion.

Localisation of an endonuclease specific for double-stranded RNA within the nucleolus and its implication in processing ribosomal transcripts.

Nucleoli of both chick embryos and mouse Ehrlich ascites cells contain an enzymatic activity that is very similar to RNase DII, an enzyme isolated from total chick embryos for its ability to degrade double-stranded RNA. The enzyme can be extracted by low salt/EDTA from nucleoli and is associated with pre-ribosomal 80-S and 55-S particles. Under ionic conditions which are inhibitory for the nucleolytic activity the transcript in vitro of nucleoli is not processed and sediments around 45 S. Under salt conditions which are optimal for the nucleolar enzyme the nucleolar transcripts are cleaved to distinct intermediate-sized molecules. Addition of the chicken RNase DII or RNase III to the nucleolar transcription system results in a similar shift of the chain length of the RNA molecules. It is concluded that a nucleolar RNase recognizing double-stranded regions in the pre-ribosomal RNA is involved in the maturation of ribosomal RNA.