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Topic:In Situ Hybridization
In situ hybridization (ISH) is a molecular technique used to detect specific nucleic acid sequences within fixed tissues and cells. In equine research, ISH is utilized to study gene expression patterns and the localization of specific RNA or DNA sequences in horse tissues. This technique provides insights into the spatial and temporal aspects of gene activity, helping to elucidate the molecular mechanisms underlying various physiological and pathological processes in horses. ISH can be applied to a range of tissues, including those affected by diseases, to better understand the genetic contributions to equine health and disease. This page compiles peer-reviewed research studies and scholarly articles that explore the applications, methodologies, and findings of in situ hybridization in equine studies.
Factors controlling epidermal growth factor (EGF) gene expression in the endometrium of the mare. Previous studies showed a dramatic increase in EGF gene expression in the endometrial glands of pregnant mares around day 40 after ovulation. To investigate how the steroid hormones of pregnancy might regulate this expression, in situ hybridization was used to monitor the levels of EGF mRNA in endometrial biopsies obtained from seasonally anoestrous or ovariectomised mares given exogenous progesterone and oestrogen, alone or in combination, for up to 46 days. Biopsies were also taken from mares during the non-pregnant cycle, during normal pregnancies and pregnancies compromised by endometrial ...
The cDNA sequences of equine antioxidative enzyme genes Cu/Zn-SOD and Mn-SOD, and these expressions in equine tissues. The entire cDNA sequences were determined by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) techniques for equine copper/zinc superoxide dismutase (Cu/Zn-SOD) and manganese superoxide dismutase (Mn-SOD) through the use of total RNA extracted from the testis of an adult Thoroughbred. The results revealed a protein coding region for equine Cu/Zn-SOD with bases totaling 465 bp, accompanied by an estimated 154 residues of amino acids. As for equine Mn-SOD, its coding region contained a total of 669 bp and an estimated 222 residues of amino acid...
Molecular cloning of cDNA for equine follistatin and its gene expression in the reproductive tissues of the mare. A cDNA clone encoding equine follistatin was isolated from an equine ovarian cDNA library. Out of 1.2 x 10(5) independent clones screened, one positive clone was isolated and its cDNA sequence determined. The isolated clone, named EQ-FS-1, contained a complete open reading frame encoding 344 amino acid residues. The similarity of its deduced amino acid sequence to these of other mammalian species was greater than 95%. Although its expression level varied among the tissues examined, follistatin mRNA was detected in the equine uteroplacental tissues, follicles and corpora lutea by Northern blot ...
Comparison of horse chromosome 3 with donkey and human chromosomes by cross-species painting and heterologous FISH mapping. The melanocortin 1 receptor (MC1R), mast/stem cell growth factor receptor (KIT), and platelet-derived growth factor receptor alpha (PDGFRA) are loci that all belong to equine linkage group 2 (LG2). Of these, KIT was fluorescent in situ hybridization (FISH) mapped to ECA3q21 with equine cDNA and heterologous porcine BAC probes, while MC1R was localized to ECA3p12 and PDGFRA to ECA3q21 with heterologous porcine BAC probes. A three-step comparison between ECA3 and donkey chromosomes was carried out. First, microdissected ECA3 painting probe was used on donkey chromosomes, which showed disruption ...
In vitro reactivation of latent equid herpesvirus-1 from CD5+/CD8+ leukocytes indirectly by IL-2 or chorionic gonadotrophin. IL-2 and equine chorionic gonadotrophin (eCG) initiated reactivation of equid herpesvirus-1 (EHV-1) from venous lymphocytes at a frequency of 1/10(-5). Indirect immunofluorescence showed that > 80% of virus-positive leukocytes were CD5+/CD8+ with the remaining 20% being CD5+/CD8-/CD4-. Cocultivation demonstrated that the reactivated virus was infectious. In addition, virus was reactivated in vitro from leukocytes of > 70% of horses by the mitogens phytohaemagglutinin (PHA) and pokeweed mitogen (PWM). Transfer of supernatants showed that IL-2 and eCG acted indirectly by causing the releas...
Cloning and characterization of the equine F18 gene, which has a novel exon. A genomic clone isolated from an equine genomic library probed with an oligonucleotide (CAG)10 showed high sequence similarity to the human F18 gene and was tentatively named equine F18 gene. Because the human F18 gene is expressed in many tissues, we examined whether this equine clone was also expressed in equine tissues. The cDNA encoding equine F18 was obtained by the reverse transcriptase-polymerase chain reaction (RT-PCR) from equine thymus. The nucleotide sequence of the equine F18 cDNA (1940 bp) was determined and contained both the ATG initiation codon and a poly(A) sequence. The cDNA ...
Expression of inhibin alpha-subunit in horse testis. Inhibin is believed to play roles in the pituitary secretion of FSH and in the paracrine regulation of testicular function. Although it has been generally accepted that inhibin is produced in Sertoli cells, there was a recent evidence for the localization of inhibin in Leydig cells of primates, rat and sheep. However, there is no report on the expression of inhibin in the adult horse testis. Therefore, using immunohistochemistry, western blotting and in situ hybridization techniques, the present study examined inhibin alpha-subunit (Ih-alpha) expression in the adult horse testis. For the detec...
Equine infectious anemia virus is found in tissue macrophages during subclinical infection. The equine infectious anemia virus (EIAV) often results in lifelong subclinical infection following early episodes of clinical disease. To identify the cellular reservoirs of EIAV during subclinical infection, horses were infected with EIAV and allowed to develop subclinical infections. Horses with acute disease served as a basis for comparison. The tissue distribution, replication status, location of infected cells, and viral load were characterized by PCR for proviral DNA and reverse transcriptase PCR for viral RNA, in situ hybridization, and in situ PCR. Proviral DNA was widespread in tissu...
Construction of a horse BAC library and cytogenetical assignment of 20 type I and type II markers. A horse BAC library was constructed with about 40,000 clones and mean insert size of 110 kb representing a 1.5 genome equivalent coverage and a probability of finding a single sequence of 0.75. It was characterized by PCR screening of about 130 sequences of horse microsatellites and exonic gene sequences retrieved from databases. BACs containing 8 microsatellites and 12 genes were subsequently localized by fluorescent in situ hybridization (FISH) on chromosomes. Two linkage groups were newly assigned to chromosomes: LG2 to ECA3 and LG5 to ECA24, and five linkage groups were also oriented--LG3,...
Close relationship between equine and human molluscum contagiosum virus demonstrated by in situ hybridisation. To determine whether the virus responsible for human molluscum contagiosum (MCV) is the causal agent of a similar disease in horses, in situ hybridisations using cloned fragments of human MCV DNA labelled with digoxigenin were carried out on formalin-fixed biopsy sections of lesions from two horses with molluscum contagiosum-like skin lesions. In both instances there was evidence of specific hybridisation of the labelled probe to target DNA in the sections under high stringency conditions, identified by the development of a deep blue-purple stain in the cytoplasm of cells in the stratum spinos...
Expression of epidermal growth factor and its receptor in equine placental tissues. Northern blot and in situ hybridization techniques have demonstrated a marked increase in mRNA encoding epidermal growth factor (EGF) in the endometrium of mares, coincident with the start of interdigitation between the allantochorion and endometrium during placentation. In the present study, the unusually high EGF expression in the epithelium of the endometrial glands was shown to be maintained until at least day 250 of gestation (term = 320-340 days) in mares carrying normal horse conceptuses. However, in mares carrying failing donkey-in-horse pregnancies created by embryo transfer, EGF expr...
[Analysis of the distribution of ribosomal RNA genes on chromosomes of the domestic horse (Equus caballus) using fluorescent in situ hybridization]. Distribution of blocks of ribosomal RNA genes along metaphase chromosomes of 26 horses from five breeds was determined by means of a modified method of fluorescence in situ hybridization (FISH) in combination with simultaneous R- banding. Gene loci coding for rRNA were mapped to the region of secondary constriction on the short arm of chromosome 1, and to the pericentromeric regions of chromosomes 27, 28, and 31. The nucleolar organizer region (NOR) on chromosome 27 was not described earlier. Interindividual and interchromosomal NOR polymorphism was detected With the use of a semiquantitative ...
Zoo-FISH with microdissected arm specific paints for HSA2, 5, 6, 16, and 19 refines known homology with pig and horse chromosomes. Microdissected arm specific paints (ASPs) for human (HSA) chromosomes (Chrs) 2, 5, 6, 16, and 19 were used as probes on pig (SSC) and horse (ECA) metaphase chromosomes. Regions homologous to individual human arms were delineated in the two species studied. Of the ten ASPs used, HSA6 and 16 ASPs showed complete synteny conservation of individual arms as single blocks/ arms both in pig and horse. A similar trend was, in general, also observed for HSA19 ASPs. However, contrary to these observations, synteny conservation of individual arms of HSA2 and HSA5 was not observed in pig and horse. The ar...
Expression of transforming growth factor-beta 1 in normal and dyschondroplastic articular growth cartilage of the young horse. This study describes the distribution pattern of transforming growth factor-beta 1 (TGF-beta 1) mRNA and protein in normal pre- and post natal growth cartilage and alterations present in lesions of dyschondroplasia (osteochondrosis). TGF-beta 1 expression and immunoreactivity have been investigated by in situ hybridisation and immunolocalisation in the articular/epiphyseal growth cartilage of the lateral trochlear ridge of the distal femur. Cartilage was obtained from 19 normal Thoroughbred horses (5 prenatal and 14 post natal horses) and 15 post natal horses with dyschondroplasia (DCP). TGF-b...
Use of transabdominal ultrasound-guided amniocentesis for detection of equid herpesvirus 1-induced fetal infection in utero. To evaluate transabdominal ultrasound-guided amniocentesis for detection of equid herpes-virus 1 (EHV-1)-induced fetal infection in utero. Methods: 4 Welsh Mountain mares. Methods: Pregnant mares were inoculated intranasally with EHV-1 during the ninth month of gestation. Amniocentesis was initiated on postinoculation day (PID) 12, and was performed at 2- to 3-day intervals in standing mares under deep sedation. Amniotic fluid samples were tested by virus isolation (VI), polymerase chain reaction (PCR), and immunoperoxidase cytologic examination (IC) for detection of EHV-1. Results: Exposure t...
Characterization and phylogenetic significance of rhinoceros luteinizing hormone beta (LHbeta) subunit messenger RNA structure, complementary DNA sequence and gene copy number. The luteinizing hormone (LH) beta subunit gene is expressed in the pituitary glands of all mammals, whereas the closely related chorionic gonadotropin (CG) beta subunit genes have been identified only in primates and equids, and are expressed in placenta. In the case of horses, there is a single-copy equine (e) luteinizing hormone/chorionic gonadotropin hormone beta subunit gene (eLH/CGbeta) that (1) is expressed in both pituitary gland and placenta, (2) encodes a characteristic carboxyl terminal peptide (CTP) extension, and (3) transcribes an atypically elongated 5'-untranslated region (UTR) ...
Equine inhibin/activin beta A-subunit mRNA is expressed in the endometrial gland, but not in the trophoblast, during pregnancy. The expression of both inhibin alpha- and inhibin/activin beta A-subunit mRNA was examined in equine uteroplacental tissues collected during pregnancy (days 90 to 300). Northern blot analysis revealed that 5 transcripts (7.0, 4.1, 3.4, 2.6, 1.5 kb) of beta A-subunit were present, and the most abundantly expressed transcript was the 1.5 kb one. Relatively high levels of the 1.5 kb transcript were seen in the second trimester of pregnancy compared to what was found in the third trimester. To identify the tissue localization of beta A-subunit mRNA, in situ hybridization was performed, and the pos...
FISH mapping of the IGF2 gene in horse and donkey-detection of homoeology with HSA11. Three genomic subclones derived from a phage clone containing the equine IGF2 gene were used to FISH map the gene on horse (ECA) and donkey (EAS) metaphase chromosomes. The gene mapped on ECA 12q13 band and is the first locus mapped to this horse chromosome. In donkey the gene mapped very terminal on the long arm of one small submetacentric chromosome that shows almost identical DAPI-banding pattern with ECA12. This is the first locus mapped in donkey genome. Cross species chromosome painting of equine metaphase chromosomes with human Chromosome (Chr) 11-specific probe showed homoeology of thi...
Linkage of the gene for equine combined immunodeficiency disease to microsatellite markers HTG8 and HTG4; synteny and FISH mapping to ECA9. Equine combined immunodeficiency disease (CID) is caused by homozygosity for an autosomal recessive gene. To identify linked markers for the disease, we studied a family segregating for the equine CID gene. A stallion and 19 of his CID-affected offspring were tested for marker segregation at 23 microsatellite DNA loci. His CID-affected offspring inherited only one of his two alleles at the HTG8 and HTG4 loci, namely HTG8-186 and HTG4-124, respectively. Lod scores for linkage to the CID gene using a theta of 0.01 were 5.34 for HTG8 and 2.37 for HTG4. The apparent genotypes also suggested linkag...
Expression and cellular localization of inhibin alpha-subunit mRNA in equine fetal gonads. The expression of inhibin alpha-subunit mRNA in equine fetal gonads during pregnancy (Days 90 to 300) was examined by means of Northern blot analysis. In all samples examined, a single species of transcript was detected at the size of 1.5 kb. A digoxigenin-labeled antisense cRNA probe specific to equine inhibin alpha-subunit was synthesized and in situ hybridization analysis to locate the inhibin alpha-subunit mRNA positive cells was performed using frozen tissue sections of equine fetal ovary (day 150 of pregnancy) and equine fetal testis (day 180 of pregnancy). In the fetal ovary, positive c...
Expression and localization of relaxin in the ovary of the mare. Immunoreactive, chromatographic and molecular techniques were used to study the expression of relaxin in mare ovaries at different stages of the oestrous cycle. Relaxin in follicular fluid ranged from 1.6 to 2.5, from 1.4 to 5.2, from 1.2 to 6.7 and from 1.0 to 3.5 ng ml-1 in small ( 2 3 4 cm) follicles, respectively, and total content of fluid relaxin per follicle increased (P < 0.05) with follicular size. When subjected to reverse phase HPLC analysis, follicular fluid yielded absorbance profiles corresponding closely to those of purified relaxin, and immunoreactive peaks in follicular f...
Detection of latency-associated transcripts of equid herpesvirus 1 in equine leukocytes but not in trigeminal ganglia. Results from Southern hybridization and PCR amplification experiments using a randomly synthesized reverse transcription-PCR product showed that peripheral blood leukocytes from horses showing no clinical signs of disease expressed a putative latency-associated transcript antisense to and overlapping the 3' end of the equid herpesvirus 1 (EHV-1) immediate-early gene (gene 64). A PCR product derived from this transcript has > or =96% identity with the published EHV-1 sequence. In situ hybridization studies of equine bronchial lymph nodes corroborated these findings and are consistent with re...
Equine herpesvirus 4 DNA in trigeminal ganglia of naturally infected horses detected by direct in situ PCR. Neuronal and lymphoid tissues of 15 randomly selected horses were analysed post mortem by liquid nested-PCR to study the tropism of equine herpesvirus 4 (EHV-4). In four animals the trigeminal ganglia and in one case the lung were positive. Using a direct in situ PCR the EHV-4 genome was localized in the nuclei of neurons and in the bronchiolar as well as alveolar epithelium of the lung. In none of these tissues could infectious virus or viral antigens be detected. Applying the more sensitive liquid RT-PCR, however, an acute infection was demonstrated in one of the trigeminal ganglia by amplif...
Genetical and physical assignments of equine microsatellites–first integration of anchored markers in horse genome mapping. Twenty equine microsatellites were isolated from a genomic phage library, and their genetical and physical localization was sought by linkage mapping and fluorescent in situ hybridization (FISH). Nineteen of the markers were found to be polymorphic with, in most cases, heterozygosities exceeding 50%. The markers were mapped in a Swedish reference family for gene mapping, comprising eight half-sib families from Standardbred and Icelandic horse sires. Segregation was analyzed against a set of 35 other markers typed in the pedigree. Thirteen of the microsatellites showed linkage to at least one o...
Localization of the U2 linkage group of horses to ECA 3 using chromosome painting. The U2 linkage group of horses includes the genes albumin (ALB), vitamin D binding protein (GC), mitochondrial glutamate oxaloacetate transaminase 2 (GOT2), and haptoglobin (HP) which are found on two human chromosomes, namely, 4 (HSA 4) and 16 (HSA 16). Likewise these genes are also found on two different chromosomes in mice, rats, and cattle. Chromosome painting demonstrated that only horse chromosome 3 (ECA 3) hybridized with whole chromosome paints for both HSA 4 and HSA 16. This indicated that the equine U2 linkage group occurs on ECA 3, spanning the centromere. This technique will be use...
Placental localization of relaxin in the pregnant mare. In situ hybridization employing a cRNA probe derived from a 428-bp fragment of equine relaxin was used to localize relaxin mRNA, and immunocytochemistry was used to localize relaxin itself, in tissues of the placenta-endometrium interface recovered between 33 and 153 days of gestation from mares carrying intraspecific horse, interspecific mule and extraspecific donkey conceptuses. Immunocytochemical staining was also used to localize trophoblast-specific and class I major histocompatibility complex (MHC) antigens on some specimens. Relaxin mRNA and relaxin were both present in the single-cell ...
Analysis of MHC class I expression in equine trophoblast cells using in situ hybridization. Down-regulation of major histocompatibility complex (MHC) genes by trophoblast cells is considered to be a primary mechanism preventing maternal immune rejection of the fetal-placental unit in mammalian pregnancy by rendering these cells, which form the primary barrier between mother and fetus, relatively non-antigenic. In situ hybridization with probes encoding human and horse MHC class I genes was used to characterize the pattern of MHC class I mRNA expression in the various forms of horse trophoblast. Strong hybridization signals were observed in the invasive trophoblast cells of chorionic ...
Expression of types II, VI and X collagen in equine growth cartilage during development. The synthesis and expression of collagen types II, VI and X were investigated in growth cartilage selected from a group of 31 horses and ponies in the age range 157 days of gestation to 12 years. Collagen isolation, immunolocalisation and in situ hybridisation techniques were used in order to provide information on the pattern of synthesis of these 3 collagens during endochondral ossification in normal horses. Type II collagen immunoreactivity and mRNA expression was found in each of the 3 zones of growth cartilage chondrocytes in all samples studied, whereas the localisation of both collagen ...
Zoo-FISH delineates conserved chromosomal segments in horse and man. Human chromosome specific libraries (CSLs) were individually applied to equine metaphase chromosomes using the fluorescence in situ hybridization (FISH) technique. All CSLs, except Y, showed painting signals on one or several horse chromosomes. In total 43 conserved chromosomal segments were painted. Homoeology could not, however, be detected for some segments of the equine genome. This is most likely related to the very weak signals displayed by some libraries, rather than to the absence of similarity with the human genome. In spite of divergence from the human genome, dated 70-80 million yea...
