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LIBRARY _ Michigan State University PERIPHERAL BLOOD LEUCOCYTE KARYOTYPE ANAEYSIS OF TEN BREEDS 0F DOGS (CANIS FAMILIARIS) By Miriam Jean Forbes A THESIS Submitted to the College of Veterinary Medicine of Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Anatomy 196k ii ACKNOWLEDGEMENTS "Providence seldom vouschafes to mortals any more than just that degree of encour- agement which suffices to keep them at a reasonably full exertion of their powers." Certainly Hawthorne could have been referring to the research experiences of a young student. Many a student would turn away in the face of discouragement if it were not for those around him, the workers of Providence. It is indeed a pleasure, then, for the author to thank those who have helped to see this project to completion. Initial interest in the area of this research was stimulated by the authors parents, for whom she has the highest regard, both spiritually and academ- ically. Appreciation is expressed to Karl Stiles, Professor Emeritus, Department of Zoology, for his friendship and for nourishing a curiosity in the field of cytology. But the actual accomplishment of this piece of re- search is due primarily to the patient and constant en- couragement of Dr. Esther Smith, Associate Professor in the Department of Anatomy and the author's major profes- sor. Her academic interest and technical advice were invaluable. iii It is with sincere appreciation that the author thanks Dr. H. Lois Calhoun, Professor and Chairman of the Department of Anatomy, and other members of the Anatomy Department who gave so generously of their ad- vice and time. Special thanks are extended to Dr. G.B. Wilson, Professor, Department of Botany and Plant Pathology, for reading the manuscript. Appreciation is expressed to: Dr. wade 0. Brinker and Dr. walso F. Keller of the Department of Surgery and Medicine for their assistance in the procurement of blood samples. Dr. Paul Genest of the University of Laval, Quebec Canada has been a valuable colleague via the mail. His timely suggestions and donations of samples of phytohe- magglutinin tediously extracted in his lab, were greatly appreciated. The author also wishes to thank the National Science Foundation for the financial support which made the com- pletion of this thesis possible. To Turner for his patient encouragement and unlimited.faith TABLE OF CONTENTS INTRODUCTION.................................. REVIEW OF LITERATURE.......................... MATERIALS AND METHODS......................... Introduction Procurement of Samples Planting the Leucocyte Culture Harvest of Cells and Slide Preparation Karyotype Analysis RESULTS AND DISCUSSION........................ SUMMARY.AND CONCLUSIONS....................... LITERATURE CITED.............................. Page 12 12 12 13 15 18 20 28 29 LIST OF TABLES AND GRAPHS Table I. Autosome and sex chromosome counts from 13 dogs. 00.00.000.00...OOOOOOOOCOOOOO. Graph I. Growth curve for canine leucocytes in culturBOOOOOOOOOOO...OOOCOOOOOOOOOCOOOO Graph II. Effect of colchicine treatment time on the mitotic index of canine leucocytes in cultureOOOOOOOOOOOOOOOOOOOOOOOOOOOOO Graph III. Effect of colchicine concentration on mitotic index of canine leucocytes in cultureOOOOOOOOOOOOOOOOOOOOOOOOCOOOOOO vi Page 31} 35 36 37 LIST OF PLATES Page Plate IOOOOOOOOOOOOOOOOOOOOOOO0.000000000000000 38 Figure 1. Colchicine metaphase of a male dog. Figure 2. Karyogram of the chromosome complement of a male dog. Plate IIOCOCOCOOOOOOO0.000000000000000000000000 39 Figure 1. Colchicine metaphase of a fe- male dog. Figure 2. Karyogram of the chromosome complement of a female dog. Plate11100.0000000000000000000......00.0.00... “0 Figure l. Binucleate interphase cell. Figure 2. Trinucleate interphase cell. Figure 3. Quadranucleate interphase cell. Plate IV0.0.0.0....OOOOOOOOOOOCOOOOOOOOOO...0.. bl Figure 1. hN metaphase cell. Figure 2. 6N metaphase cell. Figure 3. Possible 8N metaphase cell. Plate VQOOOOOOOOOOOOCOOOCOOOOOCOOOOOOOOOOOOOO. uz Figure l. Prometaphase chromosome com- plement. Figure 2. Early metaphase chromosome complement. Figure 3. Midmetaphase chromosome com- plement. Figure A. Post-metaphase chromosome complement. '11 INTRODUCTION Although the chromosome complement has been recog- nized as the means for genetic reproduction for several decades, it has been only within the past ten years that precise descriptive studies of karyotypes of man and oth- er animals have been made. As a result of improved techniques for studying chromosome morphology, several areas of research have been given impetus into new and exciting phases. The study of "taxonomic" karyography has opened new vistas to the evolutionist. ‘With the normal karyotype well de~ fined, the cytologist has been able to approach the na- turally occurring or experimentally induced abnormal ka- ryotype with a valid basis for comparison. For the path- ologist, the discovery that many human abnormalities are associated with distinct chromosomal anomalies has given the first indications of the etiology of these deficien- 01680 unfortunately, the majority of descriptive mammali- an cytogenetic research has been confined to man, and experimental cytogenetic research to mice. A major prob- lem in any scientific investigation is to find the best experimental animal. Although mice are traditional ex- perimental animals, there is a discouraging similarity 2 between the twenty pairs of chromosomes of its karyotype. Extensive literature is available concerning the physiological, behavioral, genetical and anatomical va— riation in the domestic dog, Canis familiaris. 'Yet, literature concerning the cytogenetics of this mammal is noticably lacking. It has been recorded, however, that the female sex chromosome, the X, is the only non-acro- centric member of the karyotype. Such a characteristic would facilitate diagnostic analysis involving sex chro- mosome abnormalities. It is the purpose of this presentation to describe adequately the normal karyotpe of a spectrum of breeds of Canis familiaris, as exhibited by peripheral blood leucocyte cultures. This may establish a basis for path- ologic and experimental research and perhaps ultimately for clinical diagnostic use. REVIEW OF LITERATURE Moorhead (1962) defined the stock in trade of the cytogeneticist as the "correlation between the cytologic- ally visible chromosomes and the phenotypic expression." Thus, cytogenetics is an anatomical science in that it is morphological and descriptive, but deals particularly with the morphology of genetic content itself. Certain features of the interpm se chromatin have been used as markers for differentiating and identifying specific cells or particular sublines such as the Barr bodies, sex chromatin, and nuclear size and morphology (Moorhead, 1962). But the metaphase chromatin arrange- ment demonstrates most dramatically the orderly and con- sistent distribution of the total genetic code. The met- aphase chromosomes, in general, are constant in number within a species and with notable exceptions, constant within each cell of an organism (Swanson, 1961). Photographs of dividing cells at metaphase are usually used in making a karyotype analysis of an organ- ism. The chromosomes are cut out and arranged in homol- ogous pairs on the basis of such "taxonomic" features as (a) position of the centromere, (b) total length.of the chromosome, (c) relative length of the arms to the total length of the chromosome and to each other, and finally, h (d) secondary constrictions and satellites (Moorhead, 1962). Thus, a standard system of classification for a par- ticular karyotype may be developed. For man, the Denver classification (Book g§_gl., 1960) is most widely accept- ed. Patau (1960), working concurrently with the Denver group statistically evaluated the chromosome complement of man and demonstrated mathematically that the Denver classification was for the most part sound. The salient feature of both studies was that, contrary to earlier claims that each chromosome was identifiable, certain pairs of chromosomes were so similar in morphology that it was necessary to consider them as a group. In par- ticular, the X chromosome cannot be distinguished from similar sized autosomes. By thus classifying the chromosomes of an organism, the cytologist is able to recognize the normal karyotype and distinguish it from an abnormal one. Indeed, it is possible with opportune marker chromosomes to identify cell strains not only to a species level, but occasional- ly even to a particular subline (Moorhead et al., 1960). Experimentation with lower forms of life involving chromosomal anomalies has been prevalent for decades (Swanson, 1961). But only within the last ten years have chromosome counts.for many of the higher mammals been 5 known with certainty (Ford, 1960). Only with the devel- opment of simplified techniques within the last five years, however, has the field of experimental cytogen- etics with higher animals become practical (Warkany, 1963). It would be impossible to survey completely the literature which has been produced concerning the cyto- genetics of mammals. Indeed, there have been over five hundred articles on Down's syndrome (mongolism) in man (Stiles, l96h). It is pertinent, however, to illustrate the directions of research in this field by selected ex- amples. Down's syndrome, one of the first to be recognized as being associated with a chromosomal anomaly, is now believed to be of two types. The translocation type is often associated with a familial transmission by pheno- typically normal parents (Becker gt_§l., 1963). The type associated with triploidy of one of the chromosomes in the 13-15 group (Denver classification) is credited to non-disjunction either at meiosis in the parent or early in development of the individual with Down's syn- drome (Warkany, 1963). 'With the knowledge of which type is present, certain conclusions may be drawn concerning the etiology of the abnormality and mcre meaningful med- ical counsel given. By far the majority of human chromosomal anomalies 6 are associated with abnormal sex chromosomes. Such ab- normalities are often expressed phenotypically by biz- arre syndromes with various degrees of mental retarda- tion and physical anomalies, particularly affecting the genital organs (Barr, 1963; Schutt and Hayles, l96h; Scherz and Roeckel, 1963; Patau g§_gl., 1961). In ad- dition to Klinefelter's syndrome (XXI), Turner's syndrome (X0), and several types of multiple x females, cases of true hermaphroditimm and pseudohermaphroditism have been reported (Becker gt_gl., 1963; Miller, 1963; Barr, 1963; Kesaree and Woolley, 1963). Warkany (1963) reported five cases of male pseudo- hermaphrodites with normal karyotypes. One case of pseudohermaphroditism that he studied, however, proved to be a mosaic with hS/XO and hB/XXXY cells or a com- bination of Turner's and Klinefelter's syndrome. Such mosaicism is not without precedent. It has been found in phenotypically normal individuals (Becker and Albert, 1963b) as well as in individuals with all of the previously mentioned syndromes(Ford, 1960; Hun- gerford, 1959; Mauer and Noe, 196M; Terresen g£_gl., 196h). Mosaicism is believed to be a result of nondisJunction during meiotic division or in early embryonic develop— ment (Ford, 1960). Carr (1963), studying human aborted fetuses, found that seven out of twenty-seven of them exhibited highly abnormal karyotypes. The literature contains several other°similar reports (Makino g§_gl., 1962; Delhanty, 1961). Carr plans also to investigate the karyotypes of parents of aborted infants for etiological study. Although leukemia is considered to be a type of malignancy, attempts are being made to associate par- ticular types of variations in karyotype with specific kinds of leukemia (Sandberg et al., 196k). Another field which has been opened to the cyto- geneticist is that of experimental production of chromo- somal anomalies. Ohnuki $13,333,. (1961) have worked with the induction of chromosomal anomalies by irradia- tion of tissues in culture. Ingalls g§_gl. (1963) are studying the effects on the karyotype of the in- Jection of chemicals in pregnant mice. The use of mice as experimental animals is con- venient since a great deal is known concerning their immunology, physiology and genetics. Yet, the simil- arity of the forty telocentric chromosomes in the mouse complement makes it difficult to distinguish the individ- ual chromosomes (Moorhead, 1962). So far, this discussion has centered primarily around mammalian cytogenetics in general. Emphasis has been placed on research concerning man since the great- est amount of literature in this field relates to this species. The species in which I am most interested is the domestic dog, Canis familiaris. Ginsburg and Slatis (1962) claimed that the use of dogs in genetic and cyto- genetic research should be explored: "the range of gen- etic variation in the dog includes the broadest biolog- ical spectra of physical, physiological, and behavioral traits, and systematic research on the biological bases of these traits has barely scratched the surface. There is no wider potential anywhere among mammals." Ford (1960) suggested that structural polymorphism of chromosomes may exist among various ethnic groups. Similarly, the developmamt of distinct breeds with highly predictable phenotypes suggest a remarkable variation in the genetic content, and perhaps chromosomal polymorphism in the dog. As a result of inbreeding to develop distinct breeds, many deleterious effects have been produced. Thus, congenital hip dysplasia is a major breeding hazard in the German Shepherd and has been recorded in thirty-six other breeds (Schnell, 1959). The anatomical appearance bred into the poodle and miniature breeds has presupposed the presence of patellar luxation (Hodgman, 1962). Many of the syndromes seen in man are also found in the dog.' Defects seen include hermaphroditism and pseudohermaphroditism (Hoskins et al., 1962), hemophil- ia (Brinkhous and Graham, 1950; Graham gt_gl., 19h9; Brown gt_gl., 1963), and leukemia (Khuen, l9h7; Brad- bury, 19h9; White, 19kt). Studies of the cytogenetics of the dog have been relativelygfew, although Makino (1951) stated that as early as 189k, vom Path studied the chromosomes of the dog and claimed the diploid number to be 6h. Malone (1918) studied spermatogenesis in the dog by testicular biopsy and drew the interesting conclusion that the fe- male had 22 chromosomes and the male 21, the sex being determined by the presence or absence of an X chromosome. Makino (1951) also cited Minouchi (1928) as first cor- rectly describing the sex constitution as X4Y, the same as in man, and the diploid number as 78. Makino (1952) also concluded that the number 78 correct. Hsu and Pomerat (1953) and Awa g§_gl. (1959) cultured canine embryonic lung and heart. Awa et a1. (1959) also studied spleen and liver cells. Upon cyto- logical study, both reaffirmed the 2N number of 78. The normal karyotype of the Beagle has been des- cribed morphologically as being composed of seventy-six telocentric autosomes and two sex chromosomes, a sub- metacentric X and a very small Y (Jacobson et al., 1963). Observing the cells of a canine venereal tumor, Ta— 10 kayama (1958) found 60 out of 1h6 cells with 60 chromo- somes, the others varying between 57 and 61. The cells were characterized by an increased (up to 1h) number of submetacentric chromosomes. Within the last few years, the culture of bone mar- row and peripheral blood leucocytes has become popular. Although these cells cannot be cultured indefinitely (Ben- der and Prescott, 1962), samples are easily obtained and short term growth provides sufficient material for karyo- type analyses. Moorhead g£_gl. (1960) described the orig- inal procedure for the culture of leukocytes in man. Since that time, many workers have presented modifications of the technique (Genest, 1963; Genest and Auger, 1963; 69- nest, l96h; Brooke, 1962; Marshall and Capon, 1961; Pun- nett gt_gl., 1962; Foft and Romero, 1963; Scherz and Lou- ro, 1963). These modifications usually involve (a) the source and type of ohytohemagglutinin, (b) the growth me- dia requirements, (c) the length of application and con- centration of colchicine, (d) the type of hypotonic sol- ution used and the length of application, and (e) fix- ation and slide preparation techniques. While it is not necessary to discuss these varia- tions in detail, it is tmportant to note, however, that all of these workers were using human peripheral blood, and their modifications are suited to the' NBC re- quirements. Cells of different organisms and indeed 11 cells of the same organism may vary greatly in their re- quirements for culture (Penso and Balducci, 1963). Study of the specific requirements for canine cells in culture has been for the most part neglected (Juday, 1960). Using modifications of the basic Moorhead proce- dure, Biggers and McFeely (1963), Humason and Sanders (1963) and Brown ‘gt_gl.,(l963), reported cultures of dog blood for observation of metaphase plates. Jacob- son gt_gl. (1963), described the normal Beagle karyo- type as a basis for investigating radiation effects on chromosome morphology. The only reported cytological study of abnormal dogs using this technique was made by Brown g£_gl. (1963). Although he described both canine hemophilia and pseudo- hermaphroditism, karyotype analyses were made only on the hemophiliacs which were found to have normal karyo- types. Hemophilia in man is generally believed to be associated with the normal karyotype also (Hellman g§_gl., 1961). Brown's study revealed the modal number of chro- mosomes to be 78 in all cases although cells were seen with chromosome numbers ranging from less than 78 to 80, and X chromosome numbers ranging from O to 2 in the fe- males, and O to 1 in the male. 12 MATERIALS AND METHODS A. Introductigg As was mentioned in the literature review, most researchers employing a peripheral blood culture tech- nique, have suited their procedures to human blood. To obtain optimum results with canine blood cells,it was necessary to run several series of control cultures from a male mongrel dog. .Optimum conditions determined by these tests will be included in the following dis- cussion. The culture and harvesting procedures were in gen- eral those of Moorhead et a1. (1960). B. Procurement of Samples Ten ml blood samples were obtained by venipunc- ture from adult dogs. The region from which the blood was withdrawn was surgically prepared by scrubbing with Liquid Germicidal Detergents as contamination was a ma- Jor hazard. Blood was drawn into a sterile heparinized 10 m1 syringe through a 20 gauge needle. As quickly as possible, the needle was removed and the blood trans- sParke Davis, Detroit, Michigan. 13 ferred into a sterile, heparinized (approximately 0.5 ml ammonium heparins per tube) 15 ml conical centrifuge ? tube for sedimentation. C. Planting the Leucocyte Culture Although some authors (Moorhead gt_gl., 1960; Ohnuki g§_gl., 1961) suggest mixing phytohemagglutinin with the blood before sedimentation to increase the ef- ficiency of red blood cell agglutination, others (Genest and Auger, 1963; Foft and Romero, 1963; Becker and A1- bert, 1963a) find this unnecessary. With dog blood, the addition of phytohemagglutinin at this point in the pro- cedure seemed to increase hemolysis. Therefore, phyto- hemagglutinin was not added at this time. The heparin- ized blood was allowed to stand at H’C for 1-3 hours until sedimentation of the erythrocytes left a layer of plasma on the top. Usually a layer of white blood cells formed a film between the red blood cells and the almost clear plasma. This film contained primarily neutrophils which are con- sidered by some to be "contaminants" as they are not be- lieved to undergo division in culture (Hastings g§_g1., 1961; Levine, 1956). Although these authors have devised ingenious mechanical techniques for removal of these spe- cific cells by utilizing their phagocytic properties, *Ammonium heparin, aqueous solution (1 m1 1000 USP gnits). American Hospital Supply Corporation, Evanston, llinois. this author found that with careful technique using a long, large gauge needle, the two to three ml of plas- ma containing the remaining leucocytes were easily re- moved leaving most of the neutrophil layer behind. To this plasma-cell suspension, 0.h m1 Phytohemagglutinin-Pn was added to stimulate mitosis. In agreement with other authors, some form of phy- tohemagglutinin was found necessary to initiate division in the leucocytes. The source did not seem critical since with the use of Brooke's (1962) simple technique, a good mitotic stimulant can be prepared. Eagle's Minimum Essential Medium (Eagle, 1959) was then added to the plasma-cell suspension in a quan- tity three to four times its volume. This medium proved most successful when an additional 0.2 mg arginine was added to each m1- Eagle's MEM according to the method of Smith (196h). A mixture of equal parts of dog and calf serum.was added to a final concentration of 20”. Anti- biotic concentration appeared to be critical, and after many trials, 50 units of penicillin and 50 micro-grams of streptomycin per ml of medium was most successful. The suspension was then inoculated into 16 mm screw cap tubes or roller tubes** in 2'ml aliquots (Foft and aDifco, Detroit, Michigan. *sBellco, Vineland, New Jersey. 15 Romero, 1963). The unsealed tubes were placed at a h5 angle in a constant flow 5% 002 in air incubator at 37 C. The cultures were left undisturbed for 72 hours be- fore any attempts were made to harvest the cells. Usu- ally one half of the cells were harvested at 72 hours and the remainder at 8h hours incubation. When tubes were used, the cells concentrated in a smaller area and grew in clumps. This produced bet- ter results than when the same suspension was inocula- ted into 2 oz. prescription bottles as suggested by Becker and Albert (1963a), Scherz and Louro (1963), and Genest and Auger (1963). D. Harvest of cells and slide prgparation 1. Colchicine pretreatment A colchicine pretreatment is prescribed by most authors for arresting mammalian cells at metaphase. With canine leucocytes, metaphase arrests were min- imal without colchicine and could be greatly increased by short application of a low concentration of colchi- cine. Colchicine in Hanks'(l9h9) BSS (Hanks and wallace, 19%9) was added to the cell suspension giving a final con- centration of 2.,g colchicine/m1 medium. . 2. Hypotonic treatment In order to produce swelling of intracellular vol- ume,hypotonic treatment was first suggested by Hsu and 16 Pomerat (1953), who used a basic salt solution without NaCl. Various authors have preference for other solu- tions which probably have little advantage one over the other. This author prefers Genest's (l96u) solution consisting of calf serum diluted 1:8 with distilled water. When the colchicine pro-treatment was completed, the vials were removed from the incubator and the sus— pension gently pipetted with a clean Pasteur pipette. Occasionally the cells adhered to the glass necessitat- ing a h minute treatment with .25% trypsin in a phos- phate buffered saline. The resulting cell suspensions were placed in a 15 m1 centrifuge tube and centrifuged. The supernatant was removed with a pipette and 5'm1 1:8 serum-distilled water solution was added. A gentle pipetting resuspen- ded the the cells in the cells in the hypotonic solution, and the tubes were incubated approximately 20 minutes at 37 C. This time allowed for incubation seemed to be critical as longer periods of treatment caused excessive breakage of cells. The cells which had settled out, were resuspended gently with a pipette and the suspension centrifuged. The supernatant was removed. 17 3. Fixation Two m1 of acetic-alcohol (1 part acetic acid to 3 parts methyl alcohol) were added carefully, although a gently breaking up of the cell pellet seemed helpful in separating the individual cells. Fixation was allowed to proceed for at least one hour. Due to the softening effect of acetic acid, it was found best to fix for short periods, and never longer than 2h hours. Fresh fixative was made for each sample of cells. The cells were then pipetted thorough- ly to break up the clumps. After centrifugation, the supernatant fixative was removed and replaced by 0.5 ml 1:1 acetic-alcohol solution and the cells were resus- pended. If the suspension appeared excessively cloudy, more of the 1:1 acetic-alcohol was added. This 1:1 acetic-alcohol was not allowed to act on the cells more than five minutes. h. Slide preparation Meanwhile, slides were placed on a plastic tray tilted at a h5 angle in the direct current of air from an electric fan. Using a Pasteur pipette, a few drops of fixed-cell suspension were placed on one end of the slide. The circulating air provided by the fan spread the cells and dried the suspension within 60 seconds. Another technique was used to prepare some of the 18 slides by placing a drop of suspension on an ice-cold slide. This was flamed and shaken gently to remove water droplets. Such a technique spread the cells bet- ter but increased the staining time from 15 minutes to six hours. 5. Staining method The stain used most frequently was 2% aceto-orceins although Feulgen was also tried. Cells were stained in freshly filtered aceto-orcein for 15 minutes, or six hours if flame-dried. They were then taken through 95% ethyl alcohol, 100% ethyl alcohol, and 1:1 ethyl alcohol- xylene for one minute each, followed by 100% xylene for five minutes. The preparations were mounted with #1 cover slips using Permount as the adhesive. The air dried preparations were usually stained immediately, although they may be stored indefinitely. E. Kagyotype analysis Slides were scanned on low power (1501) for meta- phase spreads. Chromosome counts were made with the oil immersion objective (135(1), and any characteristic mor- phological features noted. Up to 20 cells per sample were counted. For more detailed observation of partic- ular metaphases, photomicrographs were taken and karyo- nChroma Gesellschaft, Schmid and Co, Stuttgart, Germany. 19 grams were constructed. Photomicrographs for routine recordings were taken with a 35 mm Leica camera mounted by means of an Ipso adapter on a Bausch and Lamb Research microscope fitted with a 90X oil immersion apochromatic objective and il— luminated by a ribbon filament lamp. A Wratten 58 filter was inserted in the light beam. The film found most satisfactory was 35 mm High Contrast Copy'Filma develop- ed with Dektol. Negatives used for photographic enlarge- ments of metaphase spreads and interphase cells were made with 5X7 Kodalith Panchromatic films in a Bausch and Lomb Model L camera. The chromosome photographs were then cut out, paired and arranged on a white back- ground in order of decreasing size with the sex chromo- somes last. The results of the karyogram analyses are shown in Table I. *Eastman Kodak Company, Rochester, New York. 20 RESULTS AND DISCUSSION The development of a reliable culture technique proved to be the most difficult yet crucial aspect of this research investigation. The maximum length of time the leukocytes remained alive was 156 hours (See Graph 1). This is a much short- er period than the 17 days Bender and Prescott (1962) were able to maintain viable human leucocytes. This is undoubtedly due to inadequate growth medium requirements for canine tissues. Juday (1960) encountered a similar problem in maintaining viable canine mast cells. The addition of more arginine (Smith, 196h) and use of 20% serum (10% dog and 10% calf) seemed to improve viability. Needless to say, this is an area of study which merits attention. The maximum number of metaphases were obtained when the cells were harvested over the 72 to 8h hour incubation period (See Graph 1). Bender and Prescott (1962) found the maximum number of dividing cells could be obtained by harvest at three, seven, and ten days. They observed no mitoses at 17 and 21 days. Their 991.3389 however, maihtained a healthy appearance for two weeks, whereas the author seldom maintained canine cells for 21 longer than seven days before the cultures appeared moribund. By determining colchicine time and concentration effects, optimum conditions for demonstrating metaphases were provided. Graph 2 shows the effect of colchicine over varying lengths of treatment. The percentage of mitoses increased with time, but concurrently there was an increase in aberrant mitoses. Such metaphases in- clude colchicine-clumps with poorly staining, ill-de- fined chromatin. The optimum time of application should be one which would demonstrate a good percentage of metaphases, with the highest "take" of useful metaphase plates. This was four hours. By plotting treatment time against the percent of mitoses and comparing various colchicine concentrations (Graph 3), it was noted that the concentration of colchicine was not as critical as the treatment period. Twol1g of colchicine per ml medium was somewhat more effective than the higher and lower concentrations, and was the concentration gen- erally employed. A study of the karyograms was made both micro- scopically and photographically. Scoring was done on well spread metaphases, and the total number of chromo- somes as well as the number of X chromosomes were re- corded (Table 1). 22 In the early stages of this scoring, the occurrence of polyploid cells was not as rare-as might be expected. Ploidy was usually of the hN and 6N type, and in ad- dition, a large number of bi-nucleate and tri-nucleate cells were always present. Such nuclei were most often similar in size and centrally located, although nuclei of unequal size and eccentric location were not uncommon. It was first suspected that either or both of these phenomena were colchicine effects but controls harvested without colchicine also exhibited both polyploidy and binucleate cells. Frequent checks were made on 1000 cells which showed that the euploid metaphase to uni— nucleate cell ratio was no less than the polyploid meta- phase to multinucleate cell ratio. It is important to consider some conditions which could account for this multinucleation. Recall that (a) the cells were grown best in clumps, (b) they were fixed as a pellet, and (c) they were usually gently pipetted free. In addition, the acetic acid softened the cell membrane which became ill-defined when the slides were prepared. Therefore, the binucleate and trinucle- ate cells could result from inadequate separation of cells due to clumping, pellet formation and softened cell membrane. Polyploidy would appear to result if such a "clump" of two or three cells were dividing 23 concurrently. If this were true, however, one would ex- pect to find an irregular pattern indicating more than one set of division.figures, rather than the regularly scattered chromosomes usually observed. Plate III shows multinucleate interphase cells and Plate IV polyploid metaphase cells. Further observations should be re- corded to determine whether a certain amount of poly- ploidy in canine leucocytes is truly such an artifact or a normal condition. Puck g£_gl. (1958) found occasion- al polyploid cells in long term cultures which otherwise maintained a constant euploid karyotype. Observation of canine chromosomes is complicated by the fact that all of the autosomes are telocentric. Pairing is a function of the total length of the arms. within the 76 autosomes, there appears to be little con- sistent variation. No secondary constrictions or satel- lites are seen. Thus, attempts at grouping into a stan- dard classification at this time appears unwise.‘ If the chromosomes are fixed at mid-metaphase, coil- ing is greatest and chromosome length, minimal. At this stage (Plate V, Figure 3), they are difficult if not im- possible to pair accurately. As the chromosomes begin to progress toward the interphase condition, the arms lengthen and pairing is more easily accomplished. Similar- ly, the early metaphase chromosome is easily paired with its mate (Plate V, Figure 2). Further, with continued 2’1 regression of the colchicine metaphase, or an early prometaphase, pairing is again difficult, as overlap- ping of arms makes their separation impossible (Plate V, Figures 2 and h). Ten breeds of dogs were represented by the 13 successfully grown cultures: 2 German Short-haired Pointers, a German Shepherd, an Italian Greyhound, a Red Bone Hound, a Toy Poodle, an Irish Setter, a Boxer, a Black and Tan Hound, a Great Dane, and three mongrels. Six were female, and the remaining 7 were male. In a1 cases, chromosome number varied around a medal count of 78. Cells were observed, however, which had chromosome complements which varied from 62 to over 200 chromosomes. The possible reasons for the existence of poly- ploidy of the hN and 6N types have been discussed. The occasional occurrence of a comfiement in the range of 117 chromosomes or a triploidy can not be explained at this time, as in no case, were triploid cells seen fre- quently enough to suggest a stem-line resulting from non-disjunction. Complements of 62 to 82 were considered to be variations of the 2N condition. Such variation was also observed by Brown et a1. (1963). Variation in sex-chromosome number was also similar to that observed by Brown et a1. (1963). Cells from fe- 25 male dogs had two submetacentric chromosomes although an occasional cell was observed with only one. In males, the rule was one submetacentric chromosome, although some cells exhibited none. An obvious cause of variation in chromosome num- bers in cells can be faulty technique (Moorhead, 1962). .Certainly there could be a great tendency for the cells to burst and chromosomes to scatter after being swelled with hypotonic solutions, softened with acid and flat- tened on a slide. Puck gt_gl. (1958) suggest another source of such variation. With highly refined culture techniques de- veloped for human tissues, they have counted 2000 mitos- es finding no chromosome number other than h6 except for an occasional polyploid. Using the same technique on various animals the samsconsistency in chromosome number was not the case. An anlysis of the 112 cells of a male opossum revealed only the number 22 (or an occasional Ah), but in culturing hamster cells, in addition to a stem line of 22 chromosomes, there were many cells with 21 or 23 chromosomes. Indeed, it was possible to pro- duce clonal stem lines of these abnormal numbers of chromosomes in culture. Considering this variable effectiveness of their culture technique, Puck et a1. (1958) noted that the 26 only cell for which a medium has been definitely defined to allow infinite growth of euploid cells is the S3 Hela clonal strain. Although he described a great degree of success with human fibroblasts in culture with this me- dium he has not been as successful with culturing such other species as the hamster. Some of the chromosomal variation seen in this study of canine tissues was likely due to such imperfect tissue culture techniques. Certain— ly before extensive experimental work may be done with canine tissues, a method must be devised to maintain euploidy in cultured cells. In culturing human peripheral blood, chromosome variation frequently occurs. If there appear to be two major complement types, mosaicism is often implicated. Minor complement types are attributed to causes already discussed. In no case studied here was there more than one major complement type, indicating a lack of mosai— cism in any of the canine tissues studied. It was noted earlier that by using particular marker chromosomes, certain cell sublines could be i- dentified. Indeed, the natural development of varia- tion in the gross morphology of genetic material pro- vides the mechanism for evolution of subspecies and even species. Although no variation has yet been found in the karyograms of persons of widely separated ethnic groups, the possibility was considered. Because of 27 this, there was reason to assume that there may be chro- mosome polymorphism in the various breeds of dogs. The variation seen in the canine phenotypes is certainly ex- pressed genetically, but variation in chromosome morphol- ogy was not recognizable in the ten breeds of this study or the four breeds of other studies made earlier (Ahmed, l9hl; Jacobson et al., 1963). Such lack of chromosomal polymorphism facilitates study of pathological conditions. Many of the chromosomal aberrations in man are as- sociated with defects in genital organs and are attrib- uted to X chromosome abnormalities. It has been dif- ficult, however, to ascertain whether the abnormal (triploid or translocation or satellite, for example) chromosome was the X or another of the similar sized autosomes. In a dog with such a congenital anomaly, it would be obvious whether the X or an autosome was in- volved. It is interesting that Takayama (1958) working with canine venereal tumor cells, found the most grossly affected chromosome to be the submetacentric one, the X. 28 SUMMAFU AND CONCLUSIONS 1. Modifications of the Moorhead g£_gl. (1960) tech- nique were described for optimum culture of canine peripheral blood cells. 2. The chromosome complement of ten breeds of dogs was described. The modal euploid 2N number was found to be 78 in all cases. 3. The chromosome morphology of all breeds studied was similar. The 76 autosomes were found to be telocentric. The male sex chromosomes were a submetacentric X and a very small telocentric Y. The.females had two sub- metacentric X chromosomes. h. Reasons for variation in chromosome number in individ- ual cells of the same organism.were discussed. 5. Although the normal complement appeared to be 78, val- id experimental work will be limited until euploid strains can be maintained in culture. Further study of medium re- quirements was indicated. 29 LITERATURE CITED Ahmed, I. A. l9hl. Cytological analysis of chromosome behavior in three breeds of dogs. Proc. Roy. Soc. Edin. Sect. B. 61:107-118. Awa, A., Sakaki, M.“&Takayama, s. 1959. An in vitro study of the somatic chromosomes in several mam- mals. Jap. Jour. 2001. 12:257. Barr, M. L. 1963. Chromosomal errors in relation to sex development. Proc. Am. Assoc. Path. and Bact. Conf. Cincinnati, Ohio. p. 26a. Becker,K. L., &Albert, A. 1963a. Modifications of methods for analysis of chromosomes of leucocytes cultured from human peripheral blood. Proc. Mayo Clinic 38:197-202. Becker, K. L.,&Albert, A. 1963b. Familial translocation mongolism: a carrier exhibiting nonacrocentric translocation. Proc. Mayo Clinic 38:261-267. Becker, K. L., Sprague, R. G.;S’cAlbert, A. 1963. The chromosomal s ectrum of gonadal dysgenesis. Proc. Mayo Clinic 3 :h90-h96. Bender, M. A.,&Prescott, D. M. 1962. DNA synthesis and mitosis in cultures of human peripheral leukocytes. Biggers, J. D.,&McFeely, R. A. 1963. A simple method for the display of chromosomes fro: cultures of white blood cells with special reference to the ox. Nature 199:718-19. Book, J. J., Chu, B.,IFord, C. B., Fraccaro, M., Harn- den, D. 6., Han, T. C., Hungerford, D. A., Jacobs, P. A., Lejeune, J., Levan, A., Makino, 3., Puck, T. T., Robinson, A”,&Iflio, J. H.‘ 1960. A proposed standard system of nomenclature of human mitotic chromosomes. Am. Jour. Hum. Gen. 12:38h—388. Bradbury, R. H. l9h9. Observations on canine leukemia. Vet. Med. huzllé-ll7. Brinkhous, K. M.¢&Graham, J. B., 1950. Hemophilia in the female dog. Science 111:723. 30 Brooke, J.H. 1962. A simple method of preparing phyto- hemagglutinin. Chromosome Newsletter #7. Brown, R.C., Swanton M. C., & Brinkhous K. M. 1963. Ca- nine hemophilia and male pseudohermaphroditism. Lab 0 InveSto 12 6 961-967. Carr, D. H. 1963. Chromosome studies in abortuses and stillborn infants. Lancet 11:603-606. Delhanty, J.D. 1961. Triploid cells in a human embryo. Lancet 1:1286. Eagle, H. 1959. Amino acid metabolism in mammalian cell cul ures. Science 130:h32-k36. Foft, J.W., Romero, P.A. 1963. A modified laboratory technic for preparing chromosomes from human and animal peripheral blood leucocytes. Tech. Doc. Rep. No. SAM-TDR-63-1W, USAF Sch. of Aerospace Med. Ford,C.E..1960. Human cytogenetics: its present place and future possiblities. An. Journ. Hum. Gen. 12:10%-117. Genest, P. 1963. Production of a semi-purified phytohem- agglutinin (mucoprotein) of high potency for the study of chromosomes of leucocytes. Lancet 1:828. Genest P. 196%. Personal Communication. Department of Pathology, Faculty of Medicine, Laval University, Quebec, Canada. Genest, P., & Auger, C. 1963. Observations on the tech- nique for the study of human chromosomes by the culture of leucocytes from human peripheral blood. Can. Med. Ass'n. Jour. 88:302-307. Ginsburg, B.E., & Slatis, H. 1962. The use of purebred dogs in research problems of genetics. Proc. Animal Care Panel 12:151-156. Graham J. B., Buckwalter, J. A., Hartley, L. J., & Brink- hous, K. M. 19%9. Canine hemophilia. Observations on the course, clottin- anomaly, and the effect of blood transfusion. Jour. Exp. Med. 90:97-116. Hanks S. B., & wallace, R.E. l9h9. Relation of exygen and temperature in the preservation of tissues by re- fri eration. Proc. Soc. Exper. Biol. and Med. 71: 123 -1242. Hastings, J., Freedman 3., Rendon, 0., Cooper H.L. & Hirschorn, K. 1961. Culture of human white cells 31 using differential leucocyte separation. Nature 152:121A-1215. Hodgman, S.F.J. 1962. Abnormalities of possible heredita- ry origin in dogs. Vet. Rec. 79:1239-l2h2. HOSkinS H. P., LaCr01x, J. V0, & Fiayer,.Ko editors. 1 62. Canine- Medicine . Am. Vet. Publications. California. p0 212,'3770 Hsu, T. C., & Pomerat, C. M. 1953. Mammalian chromosomes ig vitro: a method for spreading the chromosomes of cells in tissue culture. Jour. Hered. ##z23. HUmason G. L. & Sanders, P. C. 1963. Culture and slide preparation of leucocytes from peripheral blood. Stain Tech. 38:338-3h0. Hungerford, D. A. 1959. The chromosome constitution of a human phenotypic intersex. Am. Jour. Hum. Gen. 11:215-236. Ingalls, T. Ingenito, E. F., & Curley, F. J. 1963. Ac- quired chromosomal anomalies induced in mice b in- ectéon of a teratogen in pregnancy. Science 1 l: 10- 12. Jacobson, C. B., Burkel, J. 8., & Telford, I. R. 1963. Leucocyte culture and chromosomal analysis of the beagle (Canis familiaris). Anat. Rec. 1H5:2H5. Jhday, J. L. 1960. Histological cytological and in vitro studies of canine mast cell tumors. Thesis, Depart- ment of Anatomy College of Veterinary Medicine, Michigan.State niversity. Kesaree N. & woolley, P. V. 1963. A phenotypic female with 99 chromosomes, presumably XXXXX. Jour. Ped. 63:1099-1103. Khuen, G. C. 19%7. Lgmfihatic leukemia with leukocytosis. No. Am. Vet. 2 : 59—H61. Levine . 1956. Magnetic tehcniques for in vitro isola- tion of leucocytes. Science 123:185-135. Makino . 1951. An Atlas of the Chromosome Numbers in male . Iowa State College Press, Ames, Iowa. m Makino, S. 1952. A contribution to the study of chromo- somes in some Asiatic mammals. Cytologia 16:288. i0 hide, M. 1962. A further study of the chromo- ' 32 somes in the Japanese. Chromosome 13:1%8-l62. dalone, T. M. 1918. Spermatogenesis of the dog. Trans. Am. Microscop. Soc. 37:97-110. Marshall, B., & Capon B. 1961. Factor stimulating cell division in cultured leucocytes. Lancet 11:103-104. Mauer, I., & Noe O. 196%. Triple stem-line chromosomal mogggcism in Down's syndrome (mongolism). Lancet 1: . Mellman.’ W. J., WOIman, I. J. Wrtzel, Ho A., Moorhead, P. S., & Qualls, D. H. 1961. A chromosomal female with hemophilia A. Blood 17:719. Miller 0. J. 1963. Sex determination sex differentia- tion and sex chromosome aberrat one in man. Acta Cytol. (Phila.) 7:1h8. Moorhead, P. S., Nowell, P.C., Mellman,‘W. J., Battips, C. M., & Hungerford, D. A. 1960. Lhromosome prep- arations of leucocytes cultured from human peri- pheral blood. Exp. Cell. Res. 20:613-616. Moorhead, P. S. 1962. Chromosome morphology as a genetic marker. In Genetic Analysis pf Mammalian Cells. Edited by‘D: J. Merchant and J. Neel, University of Michigan Press. Ohnuki, Y. Awa, A., & Pomerat, C. M. 1961. Chromosomal stud as on irradiated leukocytes in vitro. 61-10h. School of Aerospace Med. USAF Aerospace Medical Center, Brooks Air Force Base, Texas. Patau, K. 1960. The identification of individual chromo- somes especially in man. Am. Jour. Hum. Gen. 12: 250-276. Patau, K., Therman, E. Smith D.w., Inhorn, s. L., & Picken, B. F. 1961. Partial-trisomy syndromes. 1. Stgrge-Weber's disease. Am. Jour. Hum. Gen. 13:287- 29‘. Penso, G., & Balducci, D. 1963. Tissue Cultures in Bio- ;pgggal_fig§earch. Elsevier Pub. Co. New York. p. 9%. Puck, T. T. Cieciura, S. J., & Robinson, A. 1958. Genetics of somatic mammalian cells. III. Long term cultivation of euploid cells from human 33 and animal subjects. Jour. Exp. Med. 108:9h5-9S6. Punnett, T., Punnett, H. H.¢&Kaufmann, B. N. 1962. Prep- aration of a crude human leucocyte growth.factor from Phaseolus vulgaris. Lancet 1:1359-1360. Sandberg R. G., Takaaki, I., Kikuchi, Y3,&Grosswhite, L. H. 196A. Chromosomal differences among the acute leukemias. Ann. N. Y. Acad. Sci. 113:663- 716. Scherz, R. G.,&Louro, J. M. 1963. A simple method for making chromosome slides. Am. Jour. Clin. Path. Scherz, R. G.,&Roecke1, I. E. 1963. The XXXXY syndrome. Jour. Ped. 63:1093-1098. Schnell, G. B. 1959. Canine hip dysplasia. Lab. In- vest. 831178-1189. Schutt, A. J.,&Hayles, A. B. 196%. Intersex. Proc. Mayo Clinic 39:363-379. Smith, E. M. Personal Communication. June, 196M. Anat- omy Department, Michigan State University. Stiles, K. Personal Communication. June, 196M. Zoology Department, Michigan State University. Swanson, C. P. 1961. Cfitologz and Cxtogenetics. Pren- tice-Hall Inc., ew ersey. Takayama, S. E. 1958. Existence of a stem cell lineage in an infectious venereal tumor of the dog. Jap. Terresen, H., Vanden Berghe, H.,&Creemers, J. 1961;. Mosaic trisomy in a phenotypically normal moth- er of a mongol. Lancet 1:526~527. Warkany, J. 1963. Chromosome analyses in a pediatric department. Proc. Am. Ass'n. of Path. and Bact. Conf. Cincinnati, Ohio. p. 27a. White, E. G. 19%. Leukemia in the dog. Proc. Roy. Soc. 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NI TO TIC INDEX 28 26 24 22 20 Is l6 I4 I2 IO ON ¥ m 35 I I l I I ' r 48 60 72 84 96 I08 I20 I32 I44 I56 HOURS IN CULTURE, Graph.II. Effect of colchicine treatment time on . the mitotic index of canine leucocytes in culture. The solid line represents the total number of dividing cells. The dotted line represents the percentage of the total number of dividing cells which are useful for chromosome study. NITOTIO INDEX 20 IO 20 IO - THREE nouns TREATMENT 3" .05 5 I 2 3 4 .. FOUR HOURS TREA THEN T ' I\ .05 5 I 2 3 4 COLCI'IICINE CONCENTRATION (fly/MI) Graph III. Effect of colchicine concentration on mitotic index of canine leucpcytea in culture. 4o- 35 — .05 .ag/ml --- I icy/ml —-- 2 icy/ml 0' O I N (I I NITO TIC INDEX — N 0' O I I I0- O L l L l l l I 2 3 4 5 6 TREATMENT TIME IN HOURS 37 PLATE I Figure l. Colchicine metaphase of a nale dog. The x chromosome is indicated by an arrow (2.3% I). Figure 2. Iaryogram of the chromosome complement of a male dog. Constructed from the meta- phase shoun in Figure 1 (2,178 I). 38 no a M a]; to no (m M a“ M 0 on M» a M “ n A“ a“ A 0A «A on n0 n0 an an a “n a“ A an «a “A a“ A“ ~ K ' ' PLAIE II Figure 1. Colchicine metaphase of a female dog. The x chromosomes are indicated by ”rows (1,3h'0 I). Figure 2. Karyogram of the chromosome comple- ment of a female dog. Constructed from the metaphase shown in Figure 1 (1.3% I). . 39 ”use M noeenhee IA M (HI an. .n one. an no an an en e. Ga 00 no a. 00 an an e. 00 ea R. a. an 0e «A A... .' _—.— PLATE III Figure l. Binucleate interphase cell (1 661 I). Figure 2. Trinucleate interphase cell (1,830 I). Figure 3. Quadranucleate interphase cell (1,3MO x). PLATE IV Figure 1. Am metaphase cell (782 I). Figure 2. 6H metaphase cell (13’1-0 X). Figure 3. Possible 811 methphase cell (1589 X). #1 - —‘ ———-— Figure 1. Figure 2. Figure 3.111 Figure 1}. PLATE V Prometaphase chromosome complement 1 WI . Earl metaphase chromosome complement X). Midmetaphase chromosome complement (1,628 x). Post-metaphase chromosome complement (2,010 I). l+2 230.9% USE ONLY