nr3c1 null mutant zebrafish are viable and reveal DNA-binding-independent activities of the glucocorticoid receptor.

Glucocorticoids (GCs) play important roles in developmental and physiological processes through the transcriptional activity of their cognate receptor (Gr). Using CRISPR/Cas9 technology, we established a zebrafish null Gr mutant line and compared its phenotypes with wild type and a zebrafish line with partially silenced gr (gr s357/s357 ). Homozygous gr -/- larvae are morphologically inconspicuous and, in contrast to GR -/- knockout mice, viable through adulthood, although with reduced fitness and early life survival. Mutants gr -/- are fertile, but their reproductive capabilities fall at around 10 months of age, when, together with cardiac and intestinal abnormalities already visible at earlier stages, increased fat deposits are also observed. Mutants show higher levels of whole-body cortisol associated with overstimulated basal levels of crh and pomca transcripts along the HPI axis, which is unresponsive to a mechanical stressor. Transcriptional activity linked to immune response is also hampered in the gr -/- line: after intestinal damage by dextran sodium sulphate exposure, there are neither inflammatory nor anti-inflammatory cytokine gene responses, substantiating the hypothesis of a dual-action of the GC-GR complex on the immune system. Hence, the zebrafish gr mutant line appears as a useful tool to investigate Gr functions in an integrated in vivo model.

Low-cysteine alpha-keratins and corneous beta-proteins are initially formed in the regenerating tail epidermis of lizard.

During tail regeneration in lizards, the stratified regenerating epidermis progressively gives rise to neogenic scales that form a new epidermal generation. Initially, a soft, un-scaled, pliable, and extensible epidermis is formed that is progressively replaced by a resistant but non-extensible scaled epidermis. This suggests that the initial corneous proteins are later replaced with harder corneous proteins. Using PCR and immunocytochemistry, the present study shows an upregulation in the synthesis of low-cysteine type I and II alpha-keratins and of corneous beta-proteins with a medium cysteine content and a low content in glycine (formerly termed beta-keratins) produced at the beginning of epidermal regeneration. Quantitative PCR indicates upregulation in the production of alpha-keratin mRNAs, particularly of type I, between normal and the thicker regenerating epidermis. PCR-data also indicate a higher upregulation for cysteine-rich corneous beta-proteins and a high but less intense upregulation of low glycine corneous protein mRNAs at the beginning of scale regeneration. Immunolabeling confirms the localization of these proteins, and in particular of beta-proteins with a medium content in cysteine initially formed in the wound epidermis and later in the differentiating corneous layers of regenerating scales. It is concluded that the wound epidermis initially contains alpha-keratins and corneous beta-proteins with a lower cysteine content than more specialized beta-proteins later formed in the mature scales. These initial corneous proteins are likely related to the pliability of the wound epidermis while more specialized alpha-keratins and beta-proteins richer in glycine and cysteine are synthesized later in the mature and inflexible scales. J. Morphol. 278:119-130, 2017. ©© 2016 Wiley Periodicals,Inc.

Molecular characterization of alpha-keratins in comparison to associated beta-proteins in soft-shelled and hard-shelled turtles produced during the process of epidermal differentiation.

The tough corneous layer in the carapace and plastron of hard-shelled turtles derives from the accumulation of keratin-associated beta-proteins (KAbetaPs, formerly called beta-keratins) while these proteins are believed to be absent in soft-shelled turtles. Our bioinformatics and molecular study has instead shown that the epidermis of the soft-shelled turtle Apalone spinifera expresses beta-proteins like or even in higher amount than in the hard-shelled turtle Pseudemys nelsoni. The analysis of a carapace cDNAs library has allowed the identification and characterization of three alpha-keratins of type I and of ten beta-proteins (beta-keratins). The acidic alpha-keratins probably combine with the basic beta-proteins but the high production of beta-proteins in A. spinifera is not prevalent over that of alpha-keratin so that their combination does not determine the formation of hard corneous material. Furthermore the presence of a proline and cisteine in the beta-sheet region of beta-proteins in A. spinifera may be unsuited to form hard masses of corneous material. The higher amount of beta-proteins over alpha-keratins instead occurs in keratinocytes of the hard and inflexible epidermis of P. nelsoni determining the deposition of hard corneous material. The study suggests that the hardness of the corneous layer derives not exclusively from the interactions between alpha-keratins with KAbetaPs but also from the different dynamic of accumulation and loss of corneocytes in the corneous layer of the hard shelled turtles where a prevalent accumulation and piling of corneocytes takes place versus the soft shelled turtle where a rapid turnover of the stratum corneum occurs.

Biomolecular identification of beta-defensin-like peptides from the skin of the soft-shelled turtle Apalone spinifera.

Numerous bacteria are frequently observed in the superficial corneocytes forming the corneous layer of the soft-shelled turtle Apalona spinifera. The resistance to bacterial penetration through the living epidermis in this turtle suggests the presence of an antimicrobial barrier, possibly derived from the presence of anti-microbial peptides in the epidermis. Four beta-defensin-like peptides, named As-BD-1 to 4, have been characterized from skin tissues using molecular and bioinformatics methods. The precursor peptides contain the beta-defensin motif with the typical cysteine localization pattern. The analysis of the expression for the four different beta-defensin-like proteins show that these molecules are expressed in the skin (epidermis and dermis) of the carapace, neck, digit, and tail but are apparently not expressed in the liver or intestine under normal conditions. These data suggest that in the skin of the soft-shelled turtle there are potential effective anti-microbial peptides against epidermal bacteria.

Distribution of specific keratin-associated beta-proteins (beta-keratins) in the epidermis of the lizard Anolis carolinensis helps to clarify the process of cornification in lepidosaurians.

The epidermis of different scales in the lizard Anolis carolinensis expresses specific keratin-associated beta-proteins (beta-keratins). In order to localize the sites of accumulation of different beta-proteins, we have utilized antibodies directed against representative members of the main families of beta-proteins, the glycine-rich (HgG5), glycine-cysteine rich (HgGC3), glycine-cysteine medium-rich (HgGC10), and cysteine-rich (HgC1) beta-proteins. Immunoblotting and immunocytochemical controls confirm the specificity of the antibodies made against these proteins. Light and ultrastructural immunocytochemistry shows that the glycine-rich protein HgG5 is present in beta-layers of different body scales but is scarce in the oberhautchen and claws, and is absent in alpha-layers and adhesive setae. The cysteine-glycine-rich protein HgGC3 is low to absent in the oberhautchen, beta-layer, and mesos-layer but increases in alpha-layers. This beta-protein is low in claws where it is likely associated with the hard alpha-keratins previously studied in this lizard. The glycine-cysteine medium-rich HgGC10 protein is low in the beta-layer, higher in alpha-layers, and in the oberhautchen. This protein forms a major component of setal proteins including those of the adhesive spatula that allow this lizard to stick on vertical surfaces. HgC1 is poorly localized in most epidermis analyzed including adhesive setae and claws and appears as a minor component of the alpha-layers. In conclusion, the present study suggests that beta- and alpha-layers of lizard epidermis represent regions with different accumulation of glycine-rich proteins (mainly for mechanical resistance and hydrophobicity in the beta-layer) or cysteine-glycine-rich proteins (for both resistance and elasticity in both alpha- and beta-layers).

The knockdown of maternal glucocorticoid receptor mRNA alters embryo development in zebrafish.

In zebrafish, ovulated oocytes contain both maternal cortisol and the mRNA for the glucocorticoid receptor (gr), which is spread as granular structures throughout the ooplasm. At 0.2 hpf, this transcript is relocated in the blastodisc area and partitioned among blastomeres. At 6-8 hpf, it is replaced by zygotic transcript. We used morpholinos to block translation of both maternal and zygotic gr transcripts, and a missplicing morpholino to block post-transcriptionally the zygotic transcript alone. Only knockdown of translation produced an increase of apoptosis and subsequent craniofacial and caudal deformities with severe malformations of neural, vascular, and visceral organs in embryos and 5-dpf larvae. Such defects were rescued with trout gr2 mRNA. Microarray analysis revealed that 114 and 37 highly expressed transcripts were up- and down-regulated, respectively, by maternal Gr protein deficiency in 5-hpf embryos. These results indicate that the maternal gr transcript and protein participate in the maternal programming of zebrafish development.

Expression analysis of steroid hormone receptor mRNAs during zebrafish embryogenesis.

We have analyzed by qRT-PCR and/or RT-PCR the abundance and degradation rate of maternal mRNAs for nine steroid hormone receptors and their possible replacement by corresponding embryonic transcripts in both ovulated oocytes and embryos of zebrafish collected at 0, 1, 2, 4, 8, 12, 24 and 48 h post-fertilization (hpf). The mRNAs encoded the nuclear receptors for progesterone (pr), androgen (ar), estrogen (er alpha, er beta 1 and er beta 2), glucocorticoids (gr), mineralocorticoids (mr) and the membrane progestin receptor-alpha and beta (mpr alpha and beta). gr mRNA was the most abundant maternal transcript in oocytes and early embryos followed by er beta 2 and ar mRNAs. They declined during the first 8 hpf, being replaced, thereafter, by the embryonic messengers. er beta 1 and mr transcript levels were low until 8 hpf, but increased steadily during embryonic transcription from 24 to 48 hpf. pr transcripts were detectable only in ovulated oocytes and at 24 and 48 hpf. At these stages, there was a slight increase of er alpha mRNA that initially was very low. mPr alpha and beta mRNAs were expressed in ovulated oocytes and faintly persisted during the first 4 hpf. There was no subsequent embryonic expression of these transcripts. The possible involvement of maternal mRNAs for glucocorticoid and sex hormone receptors in the programming of early zebrafish development is intriguing, since they mainly occur at stages in which gene replication predominates over transcription.

Identification of the 11 beta-hydroxysteroid dehydrogenase type 1 mRNA and protein in human mononuclear leukocytes.

The enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) catalyzes the interconversion between inactive 11-ketoglucocorticoids and their active 11beta-hydroxy derivatives, such as cortisol and corticosterone. We have investigated the expression of 11beta-HSD1 in freshly isolated human peripheral mononuclear leukocytes (MNL). The presence of 11beta-HSD1 mRNA was demonstrated in total RNA by RT-PCR using specific primers designed on the 4th and 5th exons of the human 11beta-HSD1 gene. Fragments of the expected size were consistently detected on agarose gels, and sequencing showed complete identity with the corresponding sequence deposited in GenBank. The occurrence of 11beta-HSD1 protein was established by Western immunoblot analysis with a specific polyclonal antibody. Enzyme oxo-reductase activity was investigated by incubating 12 samples of MNL isolated from from 8 subjects with [3H]cortisone and formation of cortisol was established only in 4 subjects (yield range: 0.15-1.3%) after acetylation and TLC, blank subtraction and correction for losses. 18beta-Glycyrrhetinic acid, an inhibitor of 11 beta-HSD1, reduced cortisol production below detection limit. Dehydrogenase activity could not be demonstrated. It is suggested that, although enzyme activity of 11beta-HSD1 in circulating MNL is low, it is apparently ready for enhancement after MNL migration to sites of inflammation.

The epidermis of scales in gecko lizards contains multiple forms of beta-keratins including basic glycine-proline-serine-rich proteins.

The epidermis of scales of gecko lizards comprises alpha- and beta-keratins. Using bidimensional electrophoresis and immunoblotting, we have characterized keratins of corneous layers of scales in geckos, especially beta-keratins in digit pad lamellae. In the latter, the formation of thin bristles (setae) allow for the adhesion and climbing vertical or inverted surfaces. alpha-Keratins of 55-66 kDa remain in the acidic and neutral range of pI, while beta-keratins of 13-18 kDa show a broader variation of pI (4-10). Some protein spots for beta-keratins correspond to previously sequenced, basic glycine-proline-serine-rich beta-keratins of 169-191 amino acids. The predicted secondary structure shows that a large part of the molecule has a random-coiled conformation, small alpha helix regions, and a central region with 2-3 strands (beta-folding). The latter, termed core-box, shows homology with feather-scale-claw keratins of birds and is involved in the formation of beta-keratin filaments. Immunolocalization of beta-keratins indicates that these proteins are mainly present in the beta-layer and oberhautchen layer, including setae. The sequenced proteins of setae form bundles of keratins that determine their elongation. This process resembles that of feather-keratin on the elongation of barbule cells in feathers. It is suggested that small proteins rich in glycine, serine, and proline evolved in reptiles and birds to reinforce the mechanical resistance of the cytokeratin cytoskeleton initially present in the epidermis of scales and feathers.

Tissue-specific transcriptional initiation and activity of steroid sulfatase complementing dehydroepiandrosterone sulfate uptake and intracrine steroid activations in human adipose tissue.

Expression analysis by reverse transcriptase (RT)-PCR indicates that human adipose tissue is not likely to perform de novo synthesis of steroid hormones from cholesterol because the mRNAs of cytochromes P450scc and P450c17, and of the steroidogenic-related proteins, steroidogenic acute regulatory protein and steroidogenic factor 1, were not detected. Instead, our data support an intracrine role of adipose tissue, in which adrenal dehydroepiandrosterone sulfate (DHEA-S), the most abundant circulating androgen in man, is selectively uptaken, desulfated, and converted into bioactive androgens and estrogens. Three organic anion-transporting polypeptides-B, -D, and -E, presumably involved in DHEA-S transmembrane transport, were demonstrated at the mRNA level. While sulfotransferase expression was not found, the occurrence of steroid sulfatase (STS), converting DHEA-S to DHEA, was established at the mRNA, protein and catalytic activity levels. The 5'-rapid amplification of cDNA ends analysis showed that STS transcription in adipose tissue is regulated by the use of two promoters which differ from the prevalent placental one. The adipose transcripts contain a distinct untranslated first exon, 0a or 0b, followed by a common partially translated exon 1b, and nine other exons that are also shared by the main placental transcript. The presence of an upstream open reading frame in the new transcript variants could lead to an N-terminal divergence restricted to the cleavable signal peptide and thus not interfering with the catalytic activity of the mature STS protein. The adipose transcripts are also present in the placenta as minor isoforms. Western blotting revealed the characteristic approximately 64 kDa band of STS in both the placenta and adipose tissue. The specific enzymatic activity of STS in adipocytes was 118 pmol/10(6) cells per hour, about 50-100 times lower than in the placenta. A similar rate of [3H] DHEA-S uptake plus desulfation was measured in preadipocytes and adipocytes, equivalent to 40-45 pmol/10(6) cells per hour. Thus, an excessive accumulation of fat may out-compete other peripheral organs that are also dependent on intracrine DHEA-S utilization, especially when the adrenal production is low or declining with aging.

Aldosterone and the conquest of land.

The sequence of the phylogenetic events that preceded the appearance of aldosterone in vertebrates is described, starting from the ancestral conversion of cytochrome P450s from oxygen detoxification to xenobiotic detoxification and synthesis of oxygenated endobiotics with useful functions in intercellular signalling, such as steroid hormones. At the end of the Silurian period [438-408 million yr ago, (Mya)], a complete set of cytochrome P450s for corticoid synthesis was presumably already available, except for mitochondrial cytochrome P450c18 or aldosterone synthase encoded by CYP11B2. This gene arose by duplication of the CYP11B gene in the sarcopterygian or lobe-finned fish/tetrapod line after its divergence from the actinopterygian or ray-finned fish line 420 Mya, but before the beginning of the colonization of land by tetrapods in the late Devonian period, around 370 Mya. The fact that aldosterone is already present in Dipnoi, which occupy an evolutionary transition between water- and air-breathing but are fully aquatic, suggests that the role of this steroid was to potentiate the corticoid response to hypoxia, rather than to prevent dehydration out of the water. In terrestrial amphibians, there is no differentiation between the secretion rates and gluco- and mineralocorticoid effects of aldosterone and corticosterone. In sauropsids, plasma aldosterone concentrations are much lower than in amphibians, but regulation of salt/water balance is dependent upon both aldosterone and corticosterone, though sometimes with opposed actions. In terrestrial mammals, aldosterone acquires a specific mineralocorticoid function, because its interaction with the mineralocorticoid receptor is protected by the coexpression of the enzyme 11beta-hydroxysteroid dehydrogenase type 2, which inactivates both cortisol and corticosterone. There is evidence that aldosterone can be also synthesized extra-adrenally in brain neurons and cardiac myocytes, which lack this protection and where the effects of aldosterone oppose those of glucocorticoids. In conclusion, the phylogenetic history of aldosterone documents the erratic progression of evolutionary changes in the course of the strenuous struggle for environmental resources and survival.

Development of a sensitive and quantitative diagnostic assay for fish nervous necrosis virus based on two-target real-time PCR.

The aim of the present work was to develop two new independent SYBR Green I-based real-time PCR assays for both detection and quantification of betanodavirus, an RNA virus that infects several species of marine teleost fish causing massive mortalities in larvae and juveniles. The assays utilized two pairs of primers targeting highly conserved regions of both the RNA molecules forming the betanodavirus genome: RNA1 encoding the RNA-dependent RNA polymerase (RdRP) and RNA2 encoding the coat protein (CP). The specificity of amplifications was monitored by the melting analysis and agarose gel electrophoresis of the amplified products. The applicability of these assays was confirmed with 21 betanodavirus strains, covering all the four main clades. In addition, a BLAST (NCBI) search with the primer sequences showed no genomic cross-reactivity with other viruses. The new assays were able to quantify concentrations of betanodavirus genes ranging from 10(1) to 10(8) copies per reaction. The intra-assay coefficients of variation (CV) of threshold cycle (Ct) values of the assays were 1.5% and 1.4% for CP and RdRP RNAs, respectively. The inter-assay CVs of Ct values were 2.3% and 2.4% for CP and RdRP RNAs, respectively. Moreover, regression analysis showed a significant correlation (R2>0.97) between genome number, as determined by real-time PCR assays and the corresponding virus titer expressed as TCID50/ml of two different betanodavirus strains propagated in cell culture. The two assays were compared with a previously established one-step RT-PCR assay and with the classical virus isolation test and found to be more sensitive. In conclusion, the developed real-time RT-PCR assays are a reliable, specific and sensitive tool for the quantitative diagnosis of betanodavirus.

Genomic organization of the CYP19b genes in the rainbow trout (Oncorhynchus mykiss Walbaum).

We report the occurrence of two CYP19b genes, namely CYP19b-I and CYP19b-II, encoding forms I and II of cytochrome P450aromB, the prevalently cerebral variant of aromatase in fish, in the nuclear genome of the rainbow trout. The CYP19b-I gene is 7.6 kbp-long, more than double the size of the known fish CYP19a and b genes, owing to the presence of three introns (1, 4 and 5) that enclose repeated sequences and are longer than 1 kbp. Unlike the CYP19a genes, but similarly to the CYP19b gene of the Nile tilapia, it contains 10, and not 9, exons, including an untranslated exon 1 (83 bp), as found also in the 5' non-coding region of mammalian CYP19 genes. The 5'-UTR is composed by exon 1 and the first 41 bp of exon 2 (150 bp), whose coding region covers the first 36 amino acid residues that incorporate the transmembrane domain. The CYP19b-II gene is only 2.5 kbp-long, because it contains only one intron, corresponding to the third intron of CYP19b-I, and lacks also its first two exons. Thus, it encodes for a presumably soluble protein. Apart from this difference, the rest of the coding region is virtually the same as that of the CYP19b-I gene. The 5'-UTR corresponds in part to the 3'-end (132 bp) of the second intron of the CYP19b-I gene, while the remaining portion (208 bp) bears no homology. CYP19b-II could be regarded as a pseudogene of the CYP19b-I gene, though it is unclear whether it is a processed or a duplicated pseudogene. Moreover, since it is transcriptionally active, it may retain a functional role for the overall brain aromatase activity in the rainbow trout.

Myostatin expression during development and chronic stress in zebrafish (Danio rerio).

Myostatin, a member of the transforming growth factor-beta superfamily, is a negative regulator of skeletal muscle mass in mammals. We have studied myostatin expression during embryonic and post-hatching development in zebrafish by semiquantitative RT-PCR. The transcript is present in just-fertilized eggs and declines at 8 h post-fertilization (hpf), suggesting a maternal origin. A secondary rise occurs at 16 hpf, indicating the onset of embryonic transcription at the time of muscle cell differentiation. The level of myostatin mRNA decreases slightly at 24 hpf, when somitogenesis is almost concluded, and rises again at and after hatching, during the period of limited muscle hyperplastic growth that is typical of slow-growing, small fish. In the adult muscle, we found the highest expression of myostatin mRNA and protein, which were detectable by Northern and Western blot analyses respectively. Although only the precursor protein form was revealed in the adult lateral muscle, we demonstrated that zebrafish myostatin is proteolytically processed and secreted in cultured cells, as is its mammalian counterpart. These results suggest that myostatin may play an important regulatory role during myogenesis and muscle growth in fish, as it does in mammals. In chronically stressed fish, grown from 16 days post-fertilization to adulthood in an overcrowded environment, we observed both depression of body growth and a diminished level of myostatin mRNA in the adult muscle, as compared with controls. We propose that chronic stunting in fish brings about a general depression of muscle protein synthesis which does not spare myostatin.

European sea bass (Dicentrarchus labrax L.) cytochrome P450arom: cDNA cloning, expression and genomic organization.

Cytochrome P450arom, a key enzyme in the hormonal steroidogenic pathway, mediates the conversion of androgens to estrogens. This work describes the molecular cloning of the cDNA encoding the European sea bass (Dicentrarchus labrax L.) cytochrome P450arom by means of reverse transcriptase and polymerase chain reaction (RT-PCR) and 5' and 3'-rapid amplification of cDNA ends (RACE) analyses. The cDNA is 1822bp in length and encodes a putative protein of 517 amino acids. Northern blot analysis revealed that the ovary expressed a transcript of about 2.2kb in size. Analysis of the deduced amino acid sequence indicated 62-86% identity with ovarian P450arom of other teleost fish, the highest identity being found with the Japanese flounder, Paralichthys olivaceous. Identity was lower (56-65%) with the P450arom forms first reported in teleost brain. Only 52% identity was observed with the corresponding fragment of the cartilaginous fish, Dasyatis sabina. RT-PCR revealed that the sea bass P450arom mRNA was also expressed, at low levels, in testis and brain. Between the 5' and 3'-untranslated terminal regions (UTR), the sea bass CYP19 gene contains eight introns. All introns conform to the GT/AG rule for RNA splicing and are inserted in exactly the same positions as those found in Oryzias latipes and the human CYP19 gene.

Hormonal steroidogenesis in liver and small intestine of the green frog, Rana esculenta L.

We have investigated the potential of autonomous hormonal steroidogenesis in liver and small intestine of male and female frogs, Rana esculenta, during the recovery phase. After incubation of mitochondrial fractions with [4-14C]cholesterol, female liver and intestine formed pregnenolone at a rate of 0.63 and 2.3 pmol/mg protein/h, respectively, whereas conversion by male organs was only c. 0.03 pmol/mg protein/h. Minced tissues preparations transformed [4-14C]pregnenolone into progesterone and 17alpha-hydroxypregnenolone, the former prevailing in the liver, the latter in the intestine. Moreover, both tissues produced 20alpha-dihydropregnenolone, 20alpha-dihydroprogesterone and dehydroepiandrosterone. From incubates with [4-14C]dehydroepiandrosterone, androstenedione and androst-5-ene-3beta, 17beta-diol were identified, the former being more abundant in the liver, the latter in the intestine. These results indicate that both liver and intestine in frog can be independent sources of hormonally active steroids in both sexes.

Sequence comparison and phylogenetic analysis of fish nodaviruses based on the coat protein gene.

We have amplified by reverse transcription-polymerase chain reaction (RT-PCR) and sequenced a 605-bp fragment covering the variable region of the coat protein gene of fish nodaviruses infecting European sea bass, Dicentrarchus labrax (n = 24), and shi drum, Umbrina cirrosa (n = 2), in the Mediterranean basin. Nine new isolates were identified and their sequences were combined with sequences in the literature to produce three different data sets. The first set, based on amino acid sequences, was used to verify the monophyly of fish nodaviruses. The second and third data sets, based on nucleic acids, were used to resolve the phylogenetic relationships between closely related fish nodaviruses. Phylogenetic analyses were performed according to the maximum parsimony and neighbor-joining methods. Our results support the monophyly of fish nodaviruses. Moreover, they confirm the subdivision of fish nodaviruses into four main clusters, in agreement with the previously suggested phylogeny of the genus Piscinodavirus, that was based on a smaller number of sequences and an alternative phylogenetic approach [14]. All the Mediterranean isolates were clustered in the group of the red-spotted grouper nervous necrosis virus and appear to have a restricted geographic distribution, except for one sequence-type (10 samples) that is widespread throughout the basin.

Detection of equine arteritis virus in semen by reverse transcriptase polymerase chain reaction-ELISA.

The reverse transcriptase polymerase chain reaction (RT-PCR) assay was used to detect Equine Arteritis Virus (EAV) in the semen of 88 horses and 2 donkeys, with neutralising antibodies against EAV, on the basis of the amplification of a 279 bp long fragment located in the viral polymerase gene. The RT-PCR assay revealed the virus at 4 TCID50/ml in cell culture and showed a greater sensitivity (54.4%) than cell culture isolation (33.3%). Moreover, the two samples of donkey semen were found positive. The cDNAs obtained from 14 samples of horse and 2 of donkey semen were sequenced. Comparing the sequence of reference strain Bucyrus, the analysed samples were 78-100% identical and showed a 84-97% nucleotide identity with Bucyrus isolate. The results demonstrate high levels of genomic heterogeneity among the extracted RNAs, but inside the fragment amplified a well-preserved region of 24 bp was found with only three mismatches in some samples, suggesting that this could be ideal as a probe for RT-PCR-ELISA. The RT-PCR-ELISA assay using the EAV 7 and 8 primer set, has proved to be sensitive, specific and above all directly applicable to semen. Additionally, the short time needed for the overall procedure makes this method suitable for diagnostic purposes.

Potential for estrogen synthesis and action in human normal and neoplastic thyroid tissues.

To investigate the potential of intracrine or paracrine estrogen synthesis and action in the human thyroid gland and thyroid tumors, the presence of the messenger ribonucleic acids (mRNAs) of both cytochrome P450 aromatase (P450arom) and estrogen receptor (ER) was investigated by RT-PCR with primers designed on the respective coding regions and Southern hybridization analysis with specific probes in neoplastic (n = 42), hyperplastic (n = 7), and adjacent histologically normal thyroid tissues (n = 33) obtained from 43 female and 7 male patients. Most thyroid tissues were positive for both mRNAs, but 2 normal and 3 neoplastic tissues were negative for P450arom mRNA only, 3 normal and 1 hyperplastic tissues were negative for ER mRNA only, and 2 normal tissues were double negative. In some patients, P450arom mRNA was absent in either the neoplastic tissue or the normal one. Single and double negative samples were relatively more frequent in men (n = 4) than in women (n = 7). All negative samples were positive for beta-actin mRNA. RT-PCR amplification and Southern blotting of promoter-specific untranslated 5'-termini revealed that the human thyroid gland and tumors mainly use the ovarian-type promoter, promoter II, for CYP19 expression. Transcripts with either exon I.4 or I.1 were present only in some samples and in very low copy number. When 18 neoplastic samples with their surrounding normal tissues were analyzed immunohistochemically, 57% of those that were positive for P450arom mRNA also had a positive immunoresponse for the corresponding protein. In the case of ER, the percentage was 58%. Immunostaining for P450arom was often particularly intense in neoplastic samples. When 3 adenomata and 1 papillary cancer were incubated with [1,2,6,7-3H]testosterone, 17beta-estradiol could be radiochemically identified with a maximal yield of 10.5 fmol/mg x h. In conclusion, the human thyroid gland appears to have the potential for both estrogen synthesis and intracrine or paracrine estrogen responsiveness, which seem to be greater in women than men and may become enhanced with the process of tumorigenesis.

Developmentally regulated expression and activity of 17alpha-hydroxylase/C-17,20-lyase cytochrome P450 in rat liver.

We have investigated the developmental pattern of expression and activity of 17alpha-hydroxylase/C-17,20-lyase cytochrome P450 (cytochrome P450c17) in the liver, stomach, duodenum, and testis of rats from day 18 of pregnancy to adulthood. In the male liver, the enzyme became detectable at birth (135 pmol/mg protein x min) at a level comparable to that in the testis (188 pmol/mg protein x min). The activity then increased dramatically, reaching a peak at 8 days (691 pmol/mg protein x min), which was more than 4-fold the testicular levels in rats of the same age or in adults. Thereafter it declined steadily, becoming undetectable from puberty onward. The hepatic peak followed a depression in testicular activity (58 pmol/mg protein x min) on day 6. Northern and immunoblot analyses showed a good temporal correlation between enzyme activity and the occurrence of P450c17 messenger RNA (mRNA) and protein. The same patterns of mRNA and protein occurrence were observed in female rat liver, indicating that the hepatic CYP17 expression is not sexually dimorphic. Sequencing confirmed a complete identity in the coding region between hepatic and gonadal mRNAs. Hepatic P450c17 mRNA, however, was 150-200 bases longer than the gonadal counterparts. No significant expression of mRNAs encoding P450scc and P450arom was observed in liver of either sex at any age. In stomach and duodenum, enzyme activity was much lower (maxima at 25 and 14 pmol/mg protein x min, respectively) than that in liver, but persisted from the time of weaning onward. It is suggested that the hepatic peak in P450c17 activity may serve to convert circulating progestogens into androgens for gonadal aromatization during Sertoli and granulosa cell proliferation.