Comparison of biochemical polymorphisms in mouflon and sheep: isoelectric differences in haemoglobins and quantitative variation of mouflon haemopexin

1. Of ten protein systems studied in mouflon (Ovis musimon), five were polymorphic (Tf, Hpx, EsA, X-protein, Cat). Electrophoretic mobilities of mouflon proteins did not differ from those of sheep. 2. Mouflon haemoglobin B and sheep haemoglobin B differed in isoelectric focusing. 3. Haemopexin levels in mouflon were determined by rocket immunoelectrophoresis. A trimodal distribution was apparent, with no haemopexin, low and high levels of the protein. The results are indicative of genetic control of haemopexin levels, one of the alleles being inactive (Hpx0).     

Haemopexin in sheep,mouflon and goat: genetic polymorphism,heterogeneity and partial characterization*

Benzidine staining of starch gels after electrophoresis of sera to which haematin was added revealed polymorphism of haemopexin in sheep, mouflon and goat. In sheep three phenotypes were observed, Hpx A, Hpx AB and Hpx B. Pedigree data support the hypothesis of codominant inheritance from a single locus by two alleles, Hpx^A and Hpx^B. Neuraminidase treatment of haemopexin preparations showed that Hpx B covered two variants, B1 and B2, thus indicating genetic control by three alleles (Hpx^A, Hpx^B1 and Hpx^B2). In sheep populations the frequency of Hpx^B is low. In mouflon, in addition to the two variants that are like those of sheep, absence of haemopexin was observed in some animals, by using starch gel electrophoresis as well as immunoelectrophoresis. In goat, three phenotypes were detected, Hpx A, Hpx AB and Hpx B, differing in migration from those of sheep. Haemopexins of the studied species are heterogeneous. Sialic acid is responsible for electrophoretic heterogeneity of sheep haemopexin. Chemical. composition (amino acid and carbohydrate), molecular weight (56 060) and N-terminal sequence (Leu-Pro-Pro-) of sheep haemopexin were also determined.     

Additional studies of sheep haemopexin: genetic control, frequencies and postnatal development

This study presents evidence that sheep haemopexin phenotypes are genetically controlled by three alleles, HpxA, HpxB1 and HpxB2, of a single autosomal locus. Frequencies of two alleles, HpxA and HpxB (HpxB encompasses two isoalleles, HpxB1 and HpxB2), were studied in eight sheep breeds in Czechoslovakia. The frequency of the HpxA allele was highest (ranging from 0.81 in Merino to 1.0 in East Friesian sheep). Qualitative and quantitative changes in haemopexin during postnatal development were studied by starch gel electrophoresis and rocket immunoelectrophoresis respectively. In electrophoresis, 1- or 2-day-old lambs had two very weak zones corresponding in mobility to two slower zones of adult animals. Later, the third more anodic zone appeared and gradually increased in intensity. In 1-month-old lambs the patterns were practically identical with those of adult animals. Using rocket immunoelectrophoresis, the level of haemopexin shortly after birth was practically zero. It rose sharply till the sixth day of life; then the level continued to rise slowly till about 1 month of age. The mean haemopexin level in adult sheep was 64.5 +/- 18.26 (SD) mg/100ml serum, ranging from 30.5 to 116.5 mg/100ml.     

Additional studies of sheep haemopexin: genetic control,frequencies and postnatal development*

Summary. This study presents evidence that sheep haemopexin phenotypes are genetically controlled by three alleles, Hpx^A, Hpx^B1 and Hpx^B2, of a single autosomal locus. Frequencies of two alleles, Hpx^A and Hpx^B (Hpx^B encompasses two isoalleles, Hpx^B1 and Hpx^B2), were studied in eight sheep breeds in Czechoslovakia. The frequency of the HpxA allele was highest (ranging from 0.81 in Merino to 1.0 in East Friesian sheep). Qualitative and quantitative changes in haemopexin during postnatal development were studied by starch gel electrophoresis and rocket immunoelectrophoresis respectively. In electrophoresis, 1- or 2-day-old lambs had two very weak zones corresponding in mobility to two slower zones of adult animals. Later, the third more anodic zone appeared and gradually increased in intensity. In 1-month-old lambs the patterns were practically identical with those of adult animals. Using rocket immunoelectrophoresis, the level of haemopexin shortly after birth was practically zero. It rose sharply till the sixth day of life; then the level continued to rise slowly till about 1 month of age. The mean haemopexin level in adult sheep was 64.5 ± 18.26 (SD) mg/100ml serum, ranging from 30.5 to 116.5 mg/100ml.     

Phenotyping of pig alpha 1B-glycoprotein (PO2) and haemopexin by 1D polyacrylamide gel electrophoresis and immunoblotting

Described is an alternative procedure for the phenotyping of pig alpha 1B-glycoprotein (PO2) and haemopexin. The procedure is based on the separation of serum samples by horizontal polyacrylamide gel electrophoresis, passive blotting onto a nitrocellulose (NC) sheet, and immunochemical detection using a mixture of a primary antibody (rabbit anti-pig alpha 1B or anti-pig haemopexin) and a peroxidase-labelled secondary antibody. Several NC copies can be obtained from a single gel and these can be developed with different monospecific antisera.     

Phenotyping of pig α1B-glycoprotein (PO2) and haemopexin by 1D polyacrylamide gel electrophoresis and immunoblotting

Summary. Described is an alternative procedure for the phenotyping of pig α₁B-glycoprotein (PO2) and haemopexin. The procedure is based on the separation of serum samples by horizontal polyacrylamide gel electrophoresis, passive blotting onto a nitrocellulose (NC) sheet, and immunochemical detection using a mixture of a primary antibody (rabbit anti-pig α₁B or anti-pig haemopexin) and a peroxidase-labelled secondary antibody. Several NC copies can be obtained from a single gel and these can be developed with different monospecific antisera.     

Haem transport to the liver by haemopexin. Receptor-mediated uptake with recycling of the protein

Rat [(59)Fe]haem-(125)I-labelled haemopexin complexes (700pmol/rat) associate rapidly and exclusively with the liver after intravenous injection into anaesthetized rats. The two isotopes exhibit different patterns of accumulation. Liver (125)I-labelled haemopexin is maximum 10min after injection (20+/-4.9pmol/g of liver) and then declines by 2h to the low values (about 3pmol/g of liver) seen after injection of the apoprotein. In contrast, [(59)Fe]haem accumulates in the liver for at least 2h. Haemopexin undergoes no extensive proteolysis during 2h of haem transport as shown by precipitation with acid (98%) and specific antiserum (92%) and by electrophoresis. Moreover, only 1-2% of the dose is located in extrahepatic tissues, and there is no significant urinary excretion of either (125)I or (59)Fe. Hepatic uptake at 10min is saturable, reaching 200pmol of haemopexin/g of liver and 350pmol of haem/g of liver at a dose of 9nmol/rat, whereas uptake of the apoprotein is 3-5% of the dose. This suggests that the interaction of haem-haemopexin with the liver is a specific receptor-mediated process. The complex probably interacts via the protein moiety, since the haem analogues mesohaem and deuterohaem do not affect association of the protein with the liver but the species of haemopexin does. Increasing amounts of protein are associated with the liver 5min after injection in the order: human>rabbit>rat, and haem uptake is consistently increased. For both rat and rabbit haemopexin saturation is reached at the same concentration of protein, i.e. 180-200pmol/g of liver, indicating that the different protein species bind to a common receptor. We propose that haemopexin transports haem to the liver by a specific receptor-mediated process and then returns to the circulation.     

Peptide-specific antibodies employed in determining the interspecies immunological cross-reactivity of haemopexin

1. Antibodies were raised in rabbits against nine peptides analogous to sequences of the human serum beta-glycoprotein haemopexin, and seven peptides were very antigenic. 2. One of these affinity-purified peptide-specific antibodies interacted with a highly conserved sequence of the haemopexin of five of the seven species tested. 3. Another antibody bound pig haemopexin even better than human haemopexin. 4. The overall, arbitrarily assessed, immunological cross-reactivity between the haemopexin of human and other species follows the order: rabbit greater than mouse greater than chicken greater than pig greater than rat greater than cow.     

Oxygen affinity of mouflon blood

Hemoglobin phenotypes of European mouflon and sheep were analyzed by isoelectric focusing; the results show that the sheep HbA migrated faster than that of other animals, while the mouflon HbA was more anodic than the sheep HbB. The oxygen dissociation curve of blood from mouflon is similar to that of sheep with HbA. The authors suggest that the mouflon should not be considered a direct ancestor of the Sardinian breed of sheep.     

Expression of the haemopexin-transport system in cultured mouse hepatoma cells. Links between haemopexin and iron metabolism.

Minimal deviation hepatoma (Hepa) cells, from the mouse hepatoma B7756, synthesize and secrete haemopexin and express both the haemopexin receptor and the membrane haem-binding protein (MHBP) associated with the receptor, making this cell line the first available for detailed study of both haemopexin metabolism and hepatic transport. The 17.5 kDa MHBP was detected in Triton X-100 extracts of Hepa cells by immunoblotting with goat anti-rabbit MHBP. Scatchard-type analysis of haem-125I-haemopexin binding at 4 degrees C revealed 35,000 receptors per cell of high affinity (Kd 17 nM). Haemopexin-mediated haem transport at 37 degrees C is saturable, having an apparent Km of 160 nM and a Vmax. of 7.5 pmol of haem/10(6) cells per h during exponential growth. Haem-transport capacity is highest in the period just before the cells enter their exponential phase of growth and slowest in stationary phase. Interestingly, haem-haemopexin serves as effectively as iron-transferrin as the sole source of iron for cell growth by Hepa cells. Furthermore, depriving Hepa cells of iron by treatment with desferrioxamine (DF) increases the number of cell-surface haemopexin receptors to 65,000 per cell and consequently increases haemopexin-mediated haem transport. The effects of DF do not appear to require protein synthesis since they are not prevented by cycloheximide. Treatment of Hepa cells with hydroxyurea, an inhibitor of the iron-requiring enzyme ribonucleotide reductase that is obligatory for DNA synthesis, enhanced haemopexin-mediated haem transport. Thus, these studies provide the first evidence for regulation of haem transport by the iron status of cells and suggest a linkage between haemopexin, iron homeostasis and cell growth.     

Comparative aspects of biochemical polymorphism in the blood of Caprinae species and their hybrids

Biochemical variation at 14 blood loci was reviewed, and specific features compared experimentally in sheep Ovis aries, mouflon Ovis musimon, goat Capra hircus, aoudad Ammotragus lervia and in 2 stillborn aoudad × goat hybrids. Variation at 3 loci was also studied in dall sheep Ovis dalli, bighorn sheep Ovis canadensis and rocky mountain goat Oreamnos americanus. Haemoglobin C production in an anaemic Hb AB mouflon and in mouflon × sheep hybrids was examined. Mouflon differ from domestic sheep in that synthesis of both Hb β^AHb β^Bchains is switched off during Hb C production. The mouflon × sheep hybrids switched off one or both chains depending on whether they had inherited sheep or mouflon Hb β chain genes. In general aoudad showed a closer affinity to goats than to sheep.     

The reactivity of the disulfide bonds of human serum haemopexin

The reactivity of the disulfide bonds of the specific haeme-binding plasma protein-human haemopexin has been studied with 2-mercaptoethanol. A molecule of haemopexin has six intrachain disulfide bridges (Takahashi et al., 1985) or which four are reactive while the remaining two can be reduced in the presence of greater than or equal to 4M urea. Disruption of the four reactive disulfide bonds in apohaemopexin abolishes the haeme binding ability. In equimolar haeme-haemopexin complex only one disulfide is reactive which suggests a large change in the tertiary structure of this protein on haeme binding.     

Genetic polymorphism of antithrombin III,haptoglobin, and haemopexin in wild and domestic European rabbits

Genetic polymorphism of European rabbit (Oryctolagus cuniculus) plasma proteins antithrombin III, haptoglobin, and haemopexin was investigated by means of isoelectric focusing in free and immobilized pH gradients followed by immunoblotting. The study of two wild and one domestic populations led to the recognition of six alleles of antithrombin III and haptoglobin, and five alleles of haemopexin.     

Hematology and serum biochemistry of European mouflon (<Emphasis Type="Italic">Ovis orientalis musimon</Emphasis>) in Croatia

The aim of the present study was to determine the reference intervals for the most commonly used hematological and biochemical parameters of European mouflon from a closed hunting ground in the eastern part of the Republic of Croatia. Blood samples were collected from 39 live, physically restrained, clinically normal European mouflon, as well as from 50 domestic sheep. The distribution of values within each parameter was determined and statistical differences in values between sexes were also determined. For each sample, 14 hematological and 18 biochemical parameters were analyzed. Hematology and biochemistry values of the European mouflon were also compared with the values of domestic sheep. In further studies, the established values might be useful for the health assessment of mouflon.     

Seasonal variation in grouping patterns of the forest-dwelling Cyprus mouflon Ovis orientalis

The seasonal variations in grouping patterns of the forest-dwelling Cyprus mouflon were examined over the annual cycle. Most of the trends observed in the size and composition of groupings agree with previous work on open-country living sheep. The main difference between Cyprus mouflon, which live in forest, and wild sheep living in open country, was in the overall group size, which is smaller in Cyprus mouflon. In fact, in their overall group size, Cyprus mouflon seem to follow the pattern of other forest-dwelling ungulates.     

“Conservation cloning” of vulnerable Esfahan mouflon (Ovis orientalis isphahanica): in vitro and in vivo studies

Among the wide range of bio-conservational strategies envisaged, recent accomplishments in the field of interspecies somatic cell nuclear transfer (iSCNT) hold considerable promise due to its unique potential to decelerate or prevent rapid loss of animal genetic resources, and even to revive extinct species. Accordingly, this study was carried out to investigate if in vitro matured and enucleated oocytes of domestic sheep could be used for interspecies conservation cloning of Esfahan mouflon (Ovis orientalis isphahanica), a vulnerable species classified by the International Union for Conservation of Nature. Cryo-banked fibroblasts of a mouflon (derived from a genome resource bank) and a domestic sheep (prepared during a recent study) were cultured in vitro and used for karyotyping. Prior to SCNT, fibroblast donor cells were serum starved for 5 days. Using the zona-free SCNT technique, in vitro matured and enucleated domestic sheep oocytes were reconstituted with nuclei donor cells of mouflon and domestic sheep, and their competencies for in vitro development to the blastocyst stage were compared. The cloned mouflon blastocysts were then surgically transferred into the uterus of the synchronized domestic sheep. Karyotype analysis confirmed that fibroblasts of the Esfahan mouflon had the correct number of diploid chromosomes (2n = 54). Evaluation of 907 activated reconstructs [Esfahan mouflon (n = 667), domestic sheep (n = 240)] revealed no significant difference in the term of blastocyst development (7.6 ± 0.5% vs. 9.3 ± 0.5%, respectively). After the transfer of 12 cloned Esfahan mouflon blastocysts to five domestic sheep recipients, two (40.0%) pregnancies were established in which both (100%) were sustained until caesarean section (days 147 and 150 of pregnancy, respectively) and culminated in the live births of cloned Esfahan mouflon lambs. However, the newborns did not survive and died soon after birth. Karyotype and genetic analyses confirmed that both clones had correct diploid chromosome number (2n = 54), and were genetically identical to each other in addition to their original cell donor. This study highlighted the importance of “conservation cloning” using closely related abundant alternate species.

     

Re-sequencing regions of the ovine Y chromosome in domestic and wild sheep reveals novel paternal haplotypes

The male-specific region of the ovine Y chromosome (MSY) remains poorly characterized, yet sequence variants from this region have the potential to reveal the wild progenitor of domestic sheep or examples of domestic and wild paternal introgression. The 5' promoter region of the sex-determining gene SRY was re-sequenced using a subset of wild sheep including bighorn ( Ovis canadensis ), thinhorn ( Ovis dalli spp.), urial ( Ovis vignei ), argali ( Ovis ammon ), mouflon ( Ovis musimon ) and domestic sheep ( Ovis aries ). Seven novel SNPs ( oY 2– oY 8) were revealed; these were polymorphic between but not within species. Re-sequencing and fragment analysis was applied to the MSY microsatellite SRYM18 . It contains a complex compound repeat structure and sequencing of three novel size fragments revealed that a pentanucleotide element remained fixed, whilst a dinucleotide element displayed variability within species. Comparison of the sequence between species revealed that urial and argali sheep grouped more closely to the mouflon and domestic breeds than the pachyceriforms (bighorn and thinhorn). SNP and microsatellite data were combined to define six previously undetected haplotypes. Analysis revealed the mouflon as the only species to share a haplotype with domestic sheep, consistent with its status as a feral domesticate that has undergone male-mediated exchange with domestic animals. A comparison of the remaining wild species and domestic sheep revealed that O. aries is free from signatures of wild sheep introgression.     

Immunochemical characterization of casein from rabbit mammary gland.

1. Excellent precipitating antibodies to rabbit recombined casein polypeptides were obtained in a sheep after 8 weeks of immunization with rabbit recombined polypeptides coupled to Sepharose-albumin. 2. The antiserum was assessed for specificity by several immunochemical techniques and was monospecific when tested against acid-precipitated casein, recombined casein and extracts of lactating rabbit mammary tissue. 3. A specific anti-casein immunoglobulin fraction was prepared by immunoadsorption of the antiserum by using Sepharose-recombined casein as immunoadsorbent. 4. The specific anti-casein immunoglobulin was used to prepare a Sepharose-anti-casein immunoadsorbent for the isolation of casein from extracts of rabbit mammary tissue.     

Reproductive potential of domestic Ovis aries for preservation of threatened Ovis orientalis isphahanica: in vitro and in vivo studies

Although the potential use of reproductive biotechnology for safeguarding of endangered wildlife species is undoubted, initial evaluation of the genetic and reproductive relationship between the endangered mammals and those closely related species is indispensable. Isfahan mouflon Ovis orientalis isphahanica is now considered as a threatened species by International Union for the Conservation of Nature. Therefore, little is known about the biology of this species. This study was carried out to investigate the possible reproductive potential of domestic sheep for ex situ conservation of the Isfahan mouflon. Somatic cell cultures were taken from ear biopsies of the wild and domestic sheep and were used for karyotype analysis. Semen samples were collected by electroejaculator from the wild and domestic rams. The spermatological characteristics of the collected semen samples were determined and used for both cryopreservation and cross-insemination of the synchronized wild and domestic female sheep. To establish a cryobank for the threatened species biomaterials, freezed samples of the somatic cells and semen were transferred to a cryotank. The result suggested that Isfahan mouflon has conserved its chromosomal integrity as previously observed and contains the same chromosomal number as the domestic sheep (2n = 54). The semen samples of both species revealed similar cryoviability (>35% gross motility postthawing). Cross-insemination of both species resulted in successful pregnancy. It was suggested that domestic sheep possesses the required biological characteristics to be considered for safeguarding of the Isfahan mouflon.     

In vivo and in vitro fertilizing capacity of cryopreserved European mouflon [Ovis gmelini musimon] spermatozoa used to restore genetically rare and isolated populations

European mouflon sheep are an endangered species of ovidae residing primarily in the mountenous habitat of the islands of Sardinia and Corsica. The purpose of this study was to assess the fertilizing capacity of cryopreserved European mouflon spermatozoa after AI in synchronized mouflon and domestic ewes and after IVF in in vitro matured mouflon and domestic ewe oocytes collected by OPU technique. Domestic ram (Ovis aries) spermatozoa served as control. Semen was collected by artificial vagina from three mouflons and three domestic rams during the breeding season and was cryopreserved. At thawing, no significant differences in sperm viability were found between the wild and the domestic species (53.1 +/- 4.6% versus 56.0 +/- 4.7%) whereas the percentage of acrosome-intact sperm was lower in mouflon (55.5 +/- 4.6%) than in ram semen (62.7 +/- 3.1%; P < 0.05). Lambing rate did not differ between synchronized mouflon and domestic ewes (5/11 versus 8/12) after 150 and 156 days of pregnancy, respectively. After two OPU sessions, 87 and 132 oocytes were collected from three hyperstimulated mouflon and three domestic ewes. Cryopreserved/thawed semen was inseminated with an endoscope into the uterus of corresponding species during the non-breeding season. The oocytes were matured and fertilized in vitro; 61/73 mouflon and 81/101 domestic ewe oocytes were found to be fertilized. From these, we obtained 6/61 and 17/81 blastocysts. After vitrification and thawing, the hatching rate showed no significant difference between mouflon and sheep blastocysts (4/6 versus 14/17). In conclusion, our data showed that cryopreserved mouflon spermatozoa can be successfully used to carry out a genuine and complete program of genetic restoration in small and isolated groups of European mouflons.