Multiple alleles of ACAN associated with chondrodysplastic dwarfism in Miniature horses.

Chondrodysplastic dwarfism in Miniature horses appeared to be a recessive genetic trait based on the occurrence of affected offspring by normal parents. Dwarf phenotypes vary and range from abnormal abortuses to viable offspring with evidence of skeletal dysplasia. A genome-wide association study implicated a region of ECA1 with dwarfism in Miniature horses. Aggrecan (ACAN) was a candidate gene in that region, and exons were sequenced to compare DNA sequences for dwarf and non-dwarf horses. Sequencing led to the discovery of variants in exons 2, 6, 7 and 15 associated with dwarfism. The four variants are identified with reference to Ecab 3.0 (GCF_002863925.1) as g.95291270del (rs1095048841), g.95284530C>T (ERP107353), g.95282140C>G (rs1095048823) and g.95257480_95257500del (rs1095048839) and designated here as D1, D2, D3* and D4 respectively. A previous study at another laboratory reported dwarfism associated with homozygosity for D3*. Homozygotes for those variants and compound heterozygotes for any combination of those variants always expressed a dwarfism phenotype. However, eight additional horses with dwarfism were found, seven of which were heterozygotes for D2, D3* or D4, suggesting the existence of additional ACAN alleles causing dwarfism. Among Miniature horses, the combined frequency of D1, D2, D3* and D4 was 0.163, suggesting a carrier rate of 26.2% for alleles causing chondrodysplastic dwarfism.

Generation of an equine biobank to be used for Functional Annotation of Animal Genomes project.

The Functional Annotation of Animal Genomes (FAANG) project aims to identify genomic regulatory elements in both sexes across multiple stages of development in domesticated animals. This study represents the first stage of the FAANG project for the horse, Equus caballus. A biobank of 80 tissue samples, two cell lines and six body fluids was created from two adult Thoroughbred mares. Ante-mortem assessments included full physical examinations, lameness, ophthalmologic and neurologic evaluations. Complete blood counts and serum biochemistries were also performed. At necropsy, in addition to tissue samples, aliquots of serum, ethylenediaminetetraacetic acid (EDTA) plasma, heparinized plasma, cerebrospinal fluid, synovial fluid, urine and microbiome samples from all regions of the gastrointestinal and urogenital tracts were collected. Epidermal keratinocytes and dermal fibroblasts were cultured from skin samples. All tissues were grossly and histologically evaluated by a board-certified veterinary pathologist. The results of the clinical and pathological evaluations identified subclinical eosinophilic and lymphocytic infiltration throughout the length of the gastrointestinal tract as well as a mild clinical lameness in both animals. Each sample was cryo-preserved in multiple ways, and nuclei were extracted from selected tissues. These samples represent the first published systemically healthy equine-specific biobank with extensive clinical phenotyping ante- and post-mortem. The tissues in the biobank are intended for community-wide use in the functional annotation of the equine genome. The use of the biobank will improve the quality of the reference annotation and allow all equine researchers to elucidate unknown genomic and epigenomic causes of disease.

Cervical Vertebral Lesions in Equine Stenotic Myelopathy.

Skeletal lesions in the articular processes of cervical vertebrae C2 to C7 were compared between Thoroughbred horses with cervical stenotic myelopathy (17 males, 2 females; age, 6-50 months) and controls (6 males, 3 females; age, 9-67 months). Lesions identified by magnetic resonance imaging occurred with an increased frequency and severity in diseased horses and were not limited to sites of spinal cord compression. Lesions involved both the articular cartilage and trabecular bone and were further characterized using micro-computed tomography and histopathology. The most common histologic lesions included osteochondrosis, osseous cyst-like structures, fibrous tissue replacement of trabecular bone, retained cartilage matrix spicules, and osteosclerosis. Osseous cyst-like structures were interpreted to be true bone cysts given they were a closed cavity with a cellular lining that separated the cyst from surrounding bone. This is the first report of bone cysts in the cervical articular processes of horses with cervical stenotic myelopathy. The morphology and distribution of the lesions provide additional support for the previously proposed pathogenesis that developmental abnormalities with likely secondary biomechanical influences on the cervical spine contribute to equine cervical stenotic myelopathy.

Structural and functional analysis of intra-articular interzone tissue in axolotl salamanders.

OBJECTIVE: Knowledge of mechanisms directing diarthrodial joint development may be useful in understanding joint pathologies and identifying new therapies. We have previously established that axolotl salamanders can fully repair large articular cartilage lesions, which may be due to the presence of an interzone-like tissue in the intra-articular space. Study objectives were to further characterize axolotl diarthrodial joint structure and determine the differentiation potential of interzone-like tissue in a skeletal microenvironment. DESIGN: Diarthrodial joint morphology and expression of aggrecan, brother of CDO (BOC), type I collagen, type II collagen, and growth/differentiation factor 5 (GDF5) were examined in femorotibial joints of sexually mature (>12 months) axolotls. Joint tissue cellularity was evaluated in individuals from 2 to 24 months of age. Chondrogenic potential of the interzone was evaluated by placing interzone-like tissue into 4 mm tibial defects. RESULTS: Cavitation reached completion in the femoroacetabular and humeroradial joints, but an interzone-like tissue was retained in the intra-articular space of distal limb joints. Joint tissue cellularity decreased to 7 months of age and then remained stable. Gene expression patterns of joint markers are broadly similar in developing mammals and mature axolotls. When interzone-like tissue was transplanted into critical size skeletal defects, an accessory joint developed within the defect site. CONCLUSIONS: These experiments indicate that mature axolotl diarthrodial joints are phenotypically similar to developing synovial joints in mammals. Generation of an accessory joint by interzone-like tissue suggests multipotent cellular differentiation potential similar to that of interzone cells in the mammalian fetus. The data support the axolotl as a novel vertebrate model for joint development and repair.

Intrinsic repair of full-thickness articular cartilage defects in the axolotl salamander.

OBJECTIVE: The ability to fully regenerate lost limbs has made the axolotl salamander (Ambystoma mexicanum) a valuable model for studies of tissue regeneration. The current experiments investigate the ability of these vertebrates to repair large articular cartilage defects and restore normal hyaline cartilage and joint structure independent of limb amputation. METHODS: Full-thickness articular cartilage defects were made by resection of the medial femoral condyle to the level of the metaphysis. At 0, 2 days, 1, 2, 3, 4, 6, 8, 12, 18, 24, 36 and 48 weeks post-surgery, the repair process was analyzed on H&E and Safranin-O stained 7 µm tissue sections. Symmetric Kullback-Leibler (SKL) divergences were used to assess proteoglycan staining intensities. Immunohistochemistry was performed for collagen types I and II. RESULTS: A fibrous "interzone-like" tissue occupies the intraarticular space of the axolotl femorotibial joint and no evidence of joint cavitation was observed. By 4 weeks post-surgery, cells within the defect site exhibited morphological similarities to those of the interzone-like tissue. At 24 weeks, joint structure and cartilaginous tissue repair were confirmed by immunohistochemistry for collagen types I and II. Quantitation of Safranin-O staining indicated restoration of proteoglycan content by 18 weeks. CONCLUSIONS: The axolotl femorotibial joint has morphological similarities to the developing mammalian diarthrodial joint. Cells in the intraarticular space may be homologous to the interzone tissue and contribute to intrinsic repair of full-thickness articular cartilage defects. Taken together, these results suggest that the axolotl may serve as a valuable model for the investigation of cellular and molecular mechanisms that achieve full articular cartilage repair.

Brother of CDO (BOC) expression in equine articular cartilage.

Brother of CDO (BOC) is a cell surface receptor that derives its name from the structurally related protein, cell adhesion molecule-related/down-regulated by oncogenes (CDO, sometimes CDON). High levels of BOC mRNA and protein expression have been described in embryonic tissues with active cell proliferation and ongoing cellular differentiation(1,2). A microarray-based screen of RNA isolated from 11 different adult equine tissues unexpectedly identified BOC as having an expression pattern restricted to articular cartilage. The objective of this study was to further investigate BOC expression in adult articular cartilage relative to other tissues. Both RT-qPCR and mRNA sequencing confirmed the microarray data. Steady state BOC mRNA levels in articular cartilage were substantially higher than in the other adult tissues tested, neonatal tendon, placenta, and whole embryo. The expression of BOC displayed a pattern of tissue specificity comparable to well established cartilage matrix protein biomarkers. BOC mRNA levels in articular cartilage increased with age, but were rapidly down-regulated when chondrocytes were enzymatically isolated from the cartilage matrix and expanded in monolayer culture. Relative expression patterns of CDO were broadly similar, but displayed lower fold change differences. A functional role in articular cartilage that involves Hedgehog signaling is suggested by the known binding affinity of BOC for all three Hedgehog ligands. These data also extend BOC and CDO biology to a post-mitotic and highly differentiated cell type within a mature tissue.

Structural annotation of equine protein-coding genes determined by mRNA sequencing.

The horse, like the majority of animal species, has a limited amount of species-specific expressed sequence data available in public databases. As a result, structural models for the majority of genes defined in the equine genome are predictions based on ab initio sequence analysis or the projection of gene structures from other mammalian species. The current study used Illumina-based sequencing of messenger RNA (RNA-seq) to help refine structural annotation of equine protein-coding genes and for a preliminary assessment of gene expression patterns. Sequencing of mRNA from eight equine tissues generated 293,758105 sequence tags of 35 bases each, equalling 10.28 gbp of total sequence data. The tag alignments represent approximately 207 × coverage of the equine mRNA transcriptome and confirmed transcriptional activity for roughly 90% of the protein-coding gene structures predicted by Ensembl and NCBI. Tag coverage was sufficient to refine the structural annotation for 11,356 of these predicted genes, while also identifying an additional 456 transcripts with exon/intron features that are not listed by either Ensembl or NCBI. Genomic locus data and intervals for the protein-coding genes predicted by the Ensembl and NCBI annotation pipelines were combined with 75,116 RNA-seq-derived transcriptional units to generate a consensus equine protein-coding gene set of 20,302 defined loci. Gene ontology annotation was used to compare the functional and structural categories of genes expressed in either a tissue-restricted pattern or broadly across all tissue samples.

A 4,103 marker integrated physical and comparative map of the horse genome.

A comprehensive second-generation whole genome radiation hybrid (RH II), cytogenetic and comparative map of the horse genome (2n = 64) has been developed using the 5000rad horse x hamster radiation hybrid panel and fluorescence in situ hybridization (FISH). The map contains 4,103 markers (3,816 RH; 1,144 FISH) assigned to all 31 pairs of autosomes and the X chromosome. The RH maps of individual chromosomes are anchored and oriented using 857 cytogenetic markers. The overall resolution of the map is one marker per 775 kilobase pairs (kb), which represents a more than five-fold improvement over the first-generation map. The RH II incorporates 920 markers shared jointly with the two recently reported meiotic maps. Consequently the two maps were aligned with the RH II maps of individual autosomes and the X chromosome. Additionally, a comparative map of the horse genome was generated by connecting 1,904 loci on the horse map with genome sequences available for eight diverse vertebrates to highlight regions of evolutionarily conserved syntenies, linkages, and chromosomal breakpoints. The integrated map thus obtained presents the most comprehensive information on the physical and comparative organization of the equine genome and will assist future assemblies of whole genome BAC fingerprint maps and the genome sequence. It will also serve as a tool to identify genes governing health, disease and performance traits in horses and assist us in understanding the evolution of the equine genome in relation to other species.

Expression of a human growth hormone (hGH) receptor isoform is predicted by tissue-specific alternative splicing of exon 3 of the hGH receptor gene transcript.

Four members of the GH gene family, human (h) GH-V, human chorionic somatomammotropin-A (hCS-A), hCS-B, and hCS-L, are expressed in the human placenta. In attempting to define the role of these hormones in placental development, we have structurally characterized the human placental GH receptor (GHR) mRNA. Human GHR cDNAs were cloned from a human placental cDNA library. Clone pGHR-P1 encompasses the entire hGHR-coding region and is identical to the previously reported liver hGHR cDNA, except for a precise deletion of the sequences corresponding to exon 3. This mRNA with an exon 3 deletion is the sole form of the hGHR mRNAs in the placental villi. A reverse transcription/polymerase chain reaction assay was used to further characterize GHR mRNA expression. Human GHR mRNA was detected in all placental tissues as well as in a wide spectrum of other tissues and cell lines. The distribution of the exon 3-retaining and exon 3-excluding isoforms of the hGHR mRNA shows distinct tissue specificity. Some tissues and cell lines contain only one of the two forms, and some contain a mixed population. Since exon 3 encodes a segment in the extracellular domain of the receptor, its alternative inclusion or exclusion may mediate critical alterations in hormone binding and physiological function.

Canine COL1A2 mutation resulting in C-terminal truncation of pro-alpha2(I) and severe osteogenesis imperfecta.

RNA and type I collagen were analyzed from cultured skin fibroblasts of a Beagle puppy with fractures consistent with type III osteogenesis imperfecta (OI). In a nonisotopic RNAse cleavage assay (NIRCA), the proband's RNA had a unique cleavage pattern in the region of COL1A2 encoding the C-propeptide. DNA sequence analyses identified a mutation in which nucleotides 3991-3994 ("CTAG") were replaced with "TGTCATTGG." The first seven bases of the inserted sequence were identical to nucleotides 4002-4008 of the normal canine COL1A2 sequence. The resulting frameshift changed 30 amino acids and introduced a premature stop codon. Reverse-transcription polymerase chain reaction (RT-PCR) with primers flanking the mutation site amplified two complementary DNA (cDNA) fragments for the proband and a single product for the control. Restriction enzyme digestions also were consistent with a heterozygous mutation in the proband. Type I procollagen labeled with [3H]proline was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Increased density of pC-alpha2(I) suggested comigration with the similarly sized pro-alpha2(I) derived from the mutant allele. Furthermore, a-chains were overhydroxylated and the ratio of alpha1(I):alpha2(I) was 3.2:1, consistent with the presence of alpha1(I) homotrimers. Analyses of COL1A2 and type I collagen were both consistent with the described heterozygous mutation affecting the pro-alpha2(I) C-propeptide and confirmed a diagnosis of OI.

Phenotypic stability of articular chondrocytes in vitro: the effects of culture models, bone morphogenetic protein 2, and serum supplementation.

Numerous in vitro culture models have been developed for the investigation of chondrocyte and cartilage biology. In this study, we investigated the stability of the chondrocytic phenotype in monolayer, aggregate, pellet, and explant culture models and assessed the effects of recombinant human bone morphogenetic protein 2 (rhBMP-2) and serum supplementation on the phenotype in each model. Phenotypic effects were assessed by analyses of procollagen type II, aggrecan, (V + C)- fibronectin, and procollagen type I messenger RNA expression. In monolayer cultures, we noted a characteristic loss of procollagen type II and induction of procollagen type I expression. The aggregate and pellet culture models supported matrix protein gene expression profiles more reflective of in vivo levels. In explant cultures, expression of matrix protein genes was consistently depressed. Treatment with rhBMP-2 significantly increased the expression of procollagen type II and aggrecan in monolayer cultures; however, other models showed comparatively little response. Similarly, serum supplementation significantly down-regulated procollagen type II and aggrecan expression in monolayer cultures but had less effect on gene expression in the other models. Serum supplementation increased procollagen type I expression in monolayer and aggregate cultures. These results suggest that the influence of exogenous BMP-2 and serum on expression of chondrocyte-specific matrix protein genes is influenced by aspects of substrate attachments, cellular morphology, and/or cytoskeletal organization. Finally, the analyses of fibronectin expression suggest that V and C region alternative splicing in chondrocytes is linked to the establishment of a three-dimensional multicellular complex.

Depletion of serum growth hormone in adult female rats by neonatal monosodium glutamate treatment without loss of female-specific hepatic enzymes P450 2d (IIC12) and steroid 5 alpha-reductase.

The sexually dimorphic profiles of pituitary GH secretion play a key role in regulating the expression of several sex-dependent and developmentally controlled P-450 enzymes in rat liver. Current models for P-450 regulation by GH, however, are primarily based on hypophysectomy and GH replacement experiments. The present study examines the effects on hepatic P-450 expression of neonatal injections of monosodium glutamate (MSG), which allows for the nonsurgical suppression of adult GH levels. Furthermore, the levels of other pituitary-dependent hormones, such as testosterone and estradiol, are largely unchanged in the MSG-treated rats. Although hypophysectomy and GH replacement experiments have previously demonstrated that expression of the female-specific hepatic enzymes P-450 2d (gene product IIC12) and steroid 5 alpha-reductase is strikingly dependent on continuous GH exposure, neither enzyme was decreased in adult female rat liver after the elimination of plasma GH (less than 2 ng/ml) by neonatal MSG treatment. Moreover, although the loss of circulating GH appears to be largely responsible for the more than 10- to 20-fold elevation of the male-specific hepatic P-450 forms 2a (gene product IIIA2) and RLM2 (gene product IIA2) in hypophysectomized female rats, no such elevation of the male-specific P-450s was observed in the GH-deficient MSG-treated female rats. In contrast, the female-predominant forms P-450j (gene product IIE1) and 3 (gene product IIA1) were both elevated in adult rat liver after neonatal MSG treatment, in agreement with earlier hypophysectomy studies and demonstrating the suppressive effects that GH can exert on expression of these P-450 forms. Thus, although MSG and hypophysectomy both produce GH depletion, the responsiveness of the hepatic P-450s to these endocrine manipulations differs, allowing for an expanded understanding of the role of GH in P-450 expression.

Human growth hormone-variant is a biologically active somatogen and lactogen.

The human GH-variant (hGH-V) gene, a member of the GH-PRL gene family, is expressed by the placenta during the second and third trimesters of gestation. The secreted hGH-V protein differs from pituitary GH (hGH-N) by only 13 amino acids. We have previously demonstrated that hGH-V can bind to both somatogen and lactogen cell surface receptors in vitro, but that the ratio of its somatogen to lactogen receptor-binding affinities is substantially higher than that of hGH-N. We now characterize the somatogen and lactogen bioactivities of hGH-V and contrast them to the bioactivity of hGH-N. Somatogen bioactivity was assayed by stimulation of weight gain in hypophysectomized rats, and lactogen bioactivity was assayed by the mitogenic response of the Nb2 lymphoma cell line. While the average increase in rat body weight in response to a fixed concentration of hormone was comparable using either hGH-V or hGH-N, the mitotic response of the lactogen-inducible Nb2 cells was significantly less for hGH-V. The comparable somatogen, but lower lactogen, bioactivity of hGH-V relative to hGH-N parallels the previously reported receptor binding profiles of the two hormones and suggests that hGH-V has the potential to perform a unique role during human gestation.

Signalling elements in the ultradian rhythm of circulating growth hormone regulating expression of sex-dependent forms of hepatic cytochrome P450.

Neonatal male rats were treated with monosodium glutamate (MSG) at either 2 or 4 mg/g BW on alternate days during the first 10 days of life. As adults, the 4 mg MSG-treated rats displayed the obesity, growth retardation, and reduced pituitary weights that typify this syndrome. These animals had no detectable plasma GH as determined from serial blood samples taken every 20 min for 8 consecutive h. Associated with this loss of circulating GH was an induction of the female-specific hepatic cytochrome P450 2d (gene IIC12) and the disappearance of the male-specific form of cytochrome P450 2c (gene IIC11). The catalytic activities of cytochrome P450 2c (i.e. androgen 16 alpha- and 2 alpha-hydroxylase), sex-dependent hexobarbital hydroxylase and total cytochrome P450 were similarly feminized. Rats exposed to the 2-mg dose of MSG were also obese, but their growth rates and pituitary sizes were not as severely affected as in the 4 mg MSG-treated rats. Circulating GH in these lower dosed males was secreted in a pulsatile pattern similar to that in normal males, except that the pulse amplitude was reduced as much as 90%. In spite of this profound decline in GH peak heights in the 2 mg MSG-treated males, liver metabolism was characteristically masculine. That is, female-specific cytochrome P450 2d remained undetectable, while the male forms of cytochrome P450 2c, 2a (gene IIIA2), and RLM2 (gene IIA2), their respective catalytic steroid hydroxylase activities, and associated sex-dependent drug-metabolizing enzymes were expressed at the level of or greater than that in normal males. Thus, while an ultradian pulse of circulating GH is necessary for the characteristically masculine profile of sex-specific forms of hepatic cytochrome P450, neither the amplitude of the secretory peaks nor their total GH content is critical, and these can be greatly reduced from the normal male levels.

Cartilage fibronectin isoforms: in search of functions for a special population of matrix glycoproteins.

Fibronectins are a part of the repertoire of matrix molecules produced by the chondrocyte in order to assemble a functional cartilage matrix. They are encoded by a single gene, but significant protein heterogeneity results from alternative RNA splicing. The population of fibronectin isofroms in adult cartilage is significantly different from fibronectins in other tissues and includes relatively high levels (20-30%) of ED-B(+) fibronectins and high levels (50-80%) of the cartilage specific (V + C)- isoform which lacks the V, III-15 and I-10 segments. Less than 4% of the fibronectins in cartilage are ED-A(+). The synthesis and accumulation of cartilage fibronectins are modulated in response to matrix pathology and to biochemical and mechanical mediators. In addition, alternative splicing patterns are altered when chondrocytes are allowed to dedifferentiate in monolayer culture such that the (V + C)- isoform is lost but the ED-A(+) isoform is reexpressed at high levels. Cartilage fibronectins have the potential to participate in cell signalling via integrin mediated pathways and to interact with other cartilage matrix macromolecules. The tissue-specific splicing pattern gives rise to a unique population of fibronectins within the cartilage. Together, this points to a critical role for cartilage fibronectins in chondrocyte cell biology and the organization of a biomechanically sound matrix. However, the precise function (or functions) of the cartilage fibronectins has (or have) not been defined. This minireview examines current information about the structure, synthesis and interactions of cartilage fibronectins. When possible, potential consequences of the inclusion of the ED-B segment or the exclusion of the V, III-15 and I-10 segments are discussed. The goal is to stimulate critical thought and discussion in the field about cartilage fibronectin isoforms, their function(s) in normal cartilage, and their role(s) in the pathogenesis of cartilage diseases.

Expression of the (V+C)- fibronectin isoform is tightly linked to the presence of a cartilaginous matrix.

Fibronectin is encoded by a single gene, but heterogeneity is introduced by alternative splicing of the pre-mRNA. An unique splice variant, designated (V+C)-, which deletes nucleotides encoding the V, III-15 and I-10 segments, has been identified in articular cartilage. In this study, a ribonuclease protection assay was used to quantitate expression of the (V+C)- isoform in eight canine cartilaginous tissues and in chondrocytes cultured as monolayers or in alginate beads. The (V+C)- fibronectin isoform was detected in all cartilaginous tissues examined, ranging from a low of 11% of steady-state fibronectin mRNA in the nucleus pulposus to 71% in the rib. An age dependent increase, from 18% in the epiphyseal cartilage of a newborn to 54% in the articular cartilage of dogs over 10 months of age, was observed. The ubiquitous presence of this isoform in cartilaginous tissues and its absence in all non-cartilaginous tissues examined to date is consistent with a very strong association of the (V+C)- fibronectin isoform with the cartilaginous phenotype. Results from a ribonuclease protection assay using a probe extending into the V region from III-14 were combined with the quantitative information about (V+C)- fibronection expression to develop an over-all profile of splicing within the V region in cartilage. Monolayer culture of articular chondrocytes altered fibronectin splicing patterns. The (V+C)- isoform was rapidly lost and ED-A(+) fibronectin was induced. Three-dimensional culture in alginate beads prevented induction of ED-A(+) fibronection, but failed to sustain expression of the (V+C)- isoform. Thus, some matrix component or structure, lost in cell culture, may be essential to maintain expression of the (V+C)- isoform. The possible relationship of changing patterns of fibronectin isoforms in cultured chondrocytes to maintenance of the differentiated phenotype is discussed.

Sex differences in the ultradian pattern of plasma growth hormone concentrations in mice.

Ultradian patterns of plasma GH concentration were determined in adult male and female mice. Serial blood samples were collected every 15 min over 8 h through surgically placed chronic indwelling right atrial catheters and assayed for GH content by an homologous radioimmunoassay. In both sexes, GH concentrations fluctuated episodically from baseline values that were often in the range of 2 micrograms/l which approached the limit of assay sensitivity, to peak values sometimes reaching 100 micrograms/l. Male mice, however, demonstrated a regular periodicity of GH peaks approximately every 2.5 h with interposed stable baseline concentrations that were significantly longer in duration than in females. The absence of extended baseline concentrations in females reduced cycle length to an average of 1.4 h and significantly increased the overall mean GH concentration. The duration, height and area of GH peaks and the average concentration between peaks were comparable in males and females. Sexual dimorphism in the ultradian patterns of serum GH concentrations have been shown in both mice and rats to regulate the expression of several sex-specific phenotypes. Comparing endogenous GH patterns in mice and rats demonstrates that males of both species have less frequent peaks than females over the same interval of time. This separation of GH peaks appears to be an essential element for the expression of masculine GH-dependent traits.

Growth hormone regulation of hepatic drug-metabolizing enzymes in the mouse.

Hepatic microsomal hexobarbital hydroxylase and aminopyrine N-demethylase activities increased in adult male mice following hypophysectomy to female-like levels, eliminating the normal sexually dimorphic pattern of these enzymes. Exogenous growth hormone replacement (0.08 I.U./100 g body weight/day) re-established the lower masculine activities only when administered subcutaneously once every 12 hr. Enzyme activities remained elevated at female-like levels when the same total dose of growth hormone was infused continuously using osmotic pumps or was injected once every 6 hr. These data suggest that, despite the reversed orientation of sex differences in hepatic drug-metabolizing enzymes between rats and mice (i.e. higher enzyme activities in female mice and male rats), the basic hormonal regulatory axis is similar in the two species. Cyclic fluctuations of systemic growth hormone concentrations masculinize kinetic parameters of hepatic hexobarbital hydroxylase and aminopyrine N-demethylase in both species. Rats and mice differ in that these similar hormonal signals lower the apparent Vmax in male mice, while markedly increasing the enzyme activities in male rats. It appears more likely, therefore, that species- and sex-specific differences in the total hepatic cytochrome P-450 isoenzyme populations produce the reversed sex-dependent pattern of hexobarbital hydroxylase and aminopyrine N-demethylase.

Corticosteroids alter the differentiated phenotype of articular chondrocytes.

Experimental evidence suggests that recommended dosages of some corticosteroids used clinically as antiinflammatory agents for treating arthropathies damage articular cartilage, but low dosages may be chondroprotective. The purpose of this study was to evaluate how different concentrations of methylprednisolone affect chondrocyte function and viability. Articular cartilage and chondrocytes were obtained from young adult horses, 1.5-3.5 years of age. Corticosteroid-induced changes in collagen expression were studied at the transcriptional level by Northern blot analyses and at the translational level by measuring [3H]-proline incorporation into [3H]-hydroxyproline. Fibronectin mRNA splicing patterns were evaluated with ribonuclease protection assays. Cytotoxicity was studied using erythrosin B dye exclusion. Steady-state levels of type II procollagen mRNA decreased without concurrent changes in type I procollagen expression as the medium methylprednisolone concentrations were increased from 1 x 10(1) to 1 x 10(8) pg/ml, dropping below 10% of control values by 1 x 10(5) pg/ml. Cytotoxicity occurred as methylprednisolone levels were increased further from 1 x 10(8) to 1 x 10(9) pg/ml. Changes in total collagen (protein) synthesis were less pronounced, but also demonstrated significant suppression between 1 x 10(4) and 1 x 10(8) pg/ml. Corticosteroid-induced changes in fibronectin isoform levels were evaluated in articular cartilage samples without in vitro culture. The cartilage-specific (V + C)(-) isoform was suppressed in both normal and inflamed joints by a single intraarticular injection (0.1 mg/kg) of methylprednisolone. Combined, these data indicate that methylprednisolone suppresses matrix protein markers of chondrocytic differentiation. Decreased and altered chondrocyte expression of matrix proteins likely contributes to the pathogenesis of corticosteroid-induced cartilage degeneration.

Expression of 3beta-hydroxysteroid dehydrogenase, cytochrome p450 17alpha-hydroxylase/17,20-lyase and cytochrome p450 aromatase enzymes in corpora lutea of diestrous and early pregnant mares.

In the pregnant mare, luteal estrogen production increases at the onset of equine chorionic gonadotropin (eCG) secretion by endometrial cups. In previous studies, we have demonstrated that eCG stimulates luteal androgen and estrogen production in pregnant mares. To further elucidate the regulation of steroidogenesis within the equine corpus luteum (CL) of pregnancy, we examined the expression of 3beta-hydroxysteroid dehydrogenase (3beta-HSD), cytochrome P450 17alpha-hydroxylase/17,20 lyase (P450(17alpha)) and cytochrome P450 aromatase (P450(arom)) in luteal tissue samples collected during diestrus (Days 7 to 10) and pregnancy before (Days 29 to 35) and after (Days 42 to 45) the onset of eCG secretion. Immunoblot analyses revealed a single protein per enzyme with molecular weights of 48 kDa (3beta-HSD), 58 kDa (P450(17alpha)) and 56 kDa (P450(arom)). Steady-state levels of 3beta-HSD were lower in luteal tissue of diestrus than pregnancy, but expression did not change during pregnancy. Steady-state expression of P450(17alpha) in CL of diestrus was not significantly different from that of pregnancy. During pregnancy, P450(17alpha) expression was significantly higher after the onset of eCG secretion. Steady-state expression of P450(arom) in CL of diestrus was not significantly different from that of pregnancy. During pregnancy, luteal expression of P450(arom) was significantly lower after the onset of eCG secretion. These data support the hypotheses that eCG has a differential effect on the expression of luteal steroidogenic enzymes, that the eCG-induced increase in luteal estrogen production is the result of an increase in available aromatizable androgen due to an increase in P450(17alpha) expression and activity, and that increased luteal estrogen production is not due to an increase in aromatase expression.