Camp. Biochem. Physiol., 1975, Vol.
5OA,pp. 263
to
266. Pergamon Press. Printed in Great Britain
RED BLOOD CELLS AND HEMOGLOBIN CONCENTRATION IN NORMAL DIPLOID AND SEVERAL TYPES OF POLYPLOID SALAMANDERS* PIERREDEPARIS,JEAN-CLAUDEBEETSCHEN ANDANDREJAYLET Laboratoire de Biologie g&&ale, Universite Paul-Sabatier, 118 route de Narbonne, 31077 ToulouseCedex, France (Received I November
1913)
Different blood parameters were examined in both diploid and polyploid adult Pieuro-
Abstract-l. deles newts. 2. In 2 n,
3 n and 4 n, the hemoglobin content and hematocrit values were very close. 3. The red blood cell counts decrease as the degree of polyploidy rises. 4. Autotetraploid and pentaploid animals do not regulate their hemoglobin values and are slightly
INTRODUCTION
diploid females (Beetschen, 1960), or by crossing female 4 n x male 2 n (Beetschen, 1962). Tetraploid animals were obtained either among the
A HOMEOSTATIC mechanism maintaining the concentration of hemoglobin for the species, in spite of triploidy and its effects on the erythrocyte size and number, has been recently described in adult intersex triploid chickens (Abdel-Hameed, 1972). Though not mentioned in that paper, such a homeostatic effect had already been studied in polyploids of the Urodele Amphibian, Pleurodeles waltlii, with respect to the number of erythrocytes (Deparis & Beetschen, 1965a), hematocrit and hemoglobin concentration (Deparis et al., 1965b). Although the red blood cell (RBC) number decreases in triploid and tetraploid salamanders, both triploid and diploid animals exhibit similar RBC packed volumes (hematocrit) and hemoglobin concentrations. We repeated such studies on several kinds of adult polyploid Pleurodeles now available in our laboratory (3 n, 4 n, 5 n), in order to check whether the regulation phenomena previously observed in triploid animals also occur in tetraploids and pentaploids. For this purpose we have in each case determined the RBC number, the concentration of hemoglobin and we have also measured the hematocrit value. With these results we have calculated the mean RBC volume, the mean corpuscular hemoglobin and the mean corpuscular hemoglobin concentration.
progeny of triploid females crossed with normal males (Beetschen, 1967), or by heating diploid eggs at the first furrow stage (autotetraploids) (Jaylet, 1972). Pentaploid animals were produced by cold treatment of the eggs of tetraploid females fertilized by normal sperm (Bee&hen, 1964). Erythrocyte counts were made with a Nageotte counting chamber, in 25 mm3 volume, after a l/400 part dilution of the blood in Marcano’s fluid. The amount of hemoglobin has been measured according to Drabkin’s method. The hematocrit value has been determined by the microhematocrit method. The following formulae were used in the calculations of: Mean Corpuscular Volume (M.C.V.) in ~3 = hematocrit % x 10 red cell count (millions/mm3)’ Mean Corpuscular Hemoglobin
(M.C.H.) in ppg =
hemoglobin (g/100 ml) red cell count (millions/mms)’ Mean
Corpuscular (M.C.H.C.)
Hemoglobin Concentration as a percentage =
hemoglobin (g/100 ml) volume of packed red cells (ml/l00 ml)’
MATERIALS AND METHODS
RESULTS
Triploid animals (Pleurodeles waltlii)were obtained either by cold treatment of fertilized eggs of normal * Supported by a CNRS grant in the field of developmental genetics in Urodele Amphibians (ERA No. 327).
The polyploidy causes a reduction in the number of RBC. Table 1 shows that the triploid erythrocytes are present in numbers which are about two-thirds of
263
264
PIERREDEPARIS,JEAN-CLAUDEBEETSCHENAND ANDR~JAYLET Table 1. Review table of the different erythrocyte constants in normal diploid individuals triploids (3 n), tetraploids (4 n), autotetraploids (4 n A) and pentaploids (5 n) of cases
R.B.C. (counts/mm3)
25 25 5 12 5
172,150~9400 122,100~5800 82,450+2100 65,800+ 5200 74,750 f 1500
NO.
Ploidy 2n 3n 4n 4nA 5n
Hematocrit 0
Hb content WOO ml)
R.B.C. volume (~3)
39.1+ 1 37.62 1 35*4&o+ 27.5 i 1 33.0* 1
10*510*2 le2rtO.4 9*7& I,7 7*1+@8 8~4+0*2
2290+ 87 30842 51 43002 250 4170t230 4418i406
(2 n),
Mean corpus- Mean corpuscular cular Hb Hb concentration 618rt24 84Ort 26 1172ri: 147 1078255 1122183
26.9 k 0.6 27.220.6 27.411.5 25-8 + 1.3 25.5& 26
& fiducial limit of the mean. amount of diploid erythrocytes, whereas tetraploid erythrocytes are in numbers which are about one-half the number of diploid erythrocytes, The amount of pentaploid RBC should be about 40 per cent of the mznber of diploid RBC, i.e. 70,000 mm3. The observed amount comes close to the theoretical number. Nevertheless, it must be pointed out that the amount of diploid RBC may vary within fairly broad limits (Deparis & Beetschen, 1965a) and theoretical values calculated from a series of animals are but an indication. In spite of this, it seems evident that the RBC from autotetraploid animals are present in amounts lower than they should be. A~totetraploid originate from several spawnings of normal diploid progenitors. The RBC number of the dipioid brothers of these autotetraploids is normal Consequently, anemia of the autotetraploids must be considered as a result of their peculiar genetic constitution. Regulation of the hematocrit and hemoglobin content is due to the increase in cell size. The mean RBC volume for triploid animals is about one-third greater than that of diploid animals. In tetraploid animals this RBG volume is about twice that of diploid animals. However, a greater variability is observed in the size of tetraploid cells as is shown by the fiducial limits of the mean. The results just shown with regard to the mean RBC volume are equally valid for the mean corpuscular hemoglobin. Finally, the mean corpuscular concentration in hemoglobin has been calculated for each ploidy. Because of its precision this constant is very interesting, It can be ascertained (Table 1) that the values obtained for the 2 n, 3 n and 4 n animals are very similar, thus showing that a regulating mechanism governs the hemoglobin concentration in 3 n and 4 n. However, autotetraploids and pentaploids are slightly anaemic. the
The histogram (Fig. 1) shows that the increase in ploidy is accompanied by a reciprocal decrease in the number of RBC, The increase in size of poly-
ploid cells counteracts the reduction in their number (Fankhauser, 1945). Therefore, the hematocrit value and hemoglobin concentration are identical for 2 n and 3 n. The slight difference between 2 IZand 3 n hematocrits is not significant for P = 0.05 (Student’s f-test for determining the difference between two averages). A regulation phenomenon still occurs in tetraploid animals, although the hematocrit value is significantly different from that of diploid animals tested in the present paper, but their hemoglobin concentration is almost the same and is very similar to that of the previously tested animals (Deparis & Beetschen, 1965a). Furthermore, the difference observed here is not si~ifi~nt for P=O*O5. The autotetraploid animals have a hematocrit and a hemoglobin content inferior to those of tetraploid animals obtained by crossing female 3 n x male 2 n. Thus, total regulation of the hemoglobin concentration does not occur in a certain type of tetraploidy and it would be interesting to find out if this same phenomenon exists in the anuran autotetraploids described by other authors (Kawamura & Nishioka, 1963). Such a comparative study should be most valuable, because the results from Bgak & Pueyo (1970) do not correspond with the same interpretation. These authors have compared diploid and natural tetraploid anurans from the genus ~dun~up~zry~us and indicated, for hemoglobin and cardiac LDH, a reduced activity or partial inactivation of genes in the tetraploid 0. americanus as compared to the diploid 0. cultripes. The ratio between the mean corpuscular hemoglobin values (MCH) of both species is: MCH americanus 4 nJMCH cultripes 2 n = l-11 ; for the mean corpuscular volume (MCV), the ratio is 4n/2 n = 1.19. The same calculation applied to our own results on PZeuradeies gives for MCH a ratio of 4 n/2 n = 1.91 and for MCV a ratio of 4 n/2 n = 1.87. These ratios are not those obtained from autotetraploid Pleurodeles for which they should be lower, but it is known that the populations of 0. americanus are polymorphic. We believe that the comparative study by Becak and Pueyo (1970) should have been done between diploid and tetraploid animals of the same species 0. americanus, of which a few diploid
Red blood cells and hemoglobin concentration in salamanders
265
I80 170 160 150 140 130
30-
I20 25woo ; 90 z
_ 80 70
d
60
G
50
$
40
E 30 z" 20 mg
IO ; 110:
Fig. 1. Different erythrocyte constants in diploid and polyploid individuals. The vertical lines represent the fiducial limit of the mean. Diploid individuals (2 n), triploids (3 n), tetraploids (4 n), autotetraploids (4 n A) and pentaploids (5 n). populations are known, and not between two different species, before a firm conclusion could be given on this problem of gene regulation. Of course, the situation may be different in anuran and urodelan species. In pentaploid Pleurodeles the regulation of hematocrit and hemoglobin content no longer takes place. It is not a question of size or weight as these were shown to be normal but, as is shown in Table 1, it is due to the mean RBC volume which is lower than it should be according to theoretical calculations. The volume of a pentaploid erythrocyte of Pleurodeles should be 5500 ~3 but in fact it is only 4418 ~3. Here also there is an important variability in size. The hemoglobin content per cell is also lower than it should be, theoretically, and is a
little different from that of the tetraploid cells. However, the size of pentaploid cells is different from that of the tetraploid ones. The calculated values show a certain heterogeneity in the size of Figure 1 the pentaploid cellular population. indicates only the average numbers. Thus, a perfect hemeostatic mechanism would seem to be incompatible with a high degree of polyploidy as shown by pentaploid animals, but still occurs in at least one category of tetraploids. REFERENCES F. (1972) Hemoglobin concentration in normal diploid and intersex triploid chickens: genetic inactivation or canalization? Science, Wash. 178, 864-865.
ABDEL-HAMEED
266
Pierre DEPARIS,JEAN-CLAUDE BEERWHENAND ANDRI’ZJAYLET
BECAKW. & PUEYOM. T. (1970) Gene regulation in the polyploid Amphibian Odontophrynus americanus. Expl cell Res. 63, 448450. BEETSCHENJ. C. (1960) Recherches sur I’hCtCroploidie expkrimentale chez un Amphibien Urodkle, PIeurodeles waltlii Michah. BUN. bioI. France Belg. 94, 12-127. BEETSCHEN J. C. (1962) Sur la descendance de femelles t&raploides croistes avec des m&les diploldes chez l’amphibien urodele Pleurodeles waltlii. C. r. hebd. Sganc. Acad. Sci., Paris 255, 3068-3070. BEETSCHEN J. C. (1964) Pentaplofdie expkrimentale chez l’amphibien urodkle Pleurodeles waltlii Michah. C. r. hebd. Siam. Acad. Sci., Paris, 258, 1641-1643. BEE~SCHENJ. C. (1967) Cinq g&&rations d’individus polyploides chez le Triton Pleurodeles waltlii Michah. C. r. Skanc. Sot. Biol., Paris 161, 93G936. DEPARIS P. & BEETSCHENJ. C. (1965a) RBsultats comparatifs de numerations globulaires faites sur le sang d’individus diploldes et polyplofdes du Triton Pleuro-
deles waltlii Michah. C. r. SPanc. Sot. Biol., Paris 159, 12241229. DEPARIS P., NOUVEL H. & BEETSCHENJ. C. (1965b) Taux d’h6moglobine et valeur de l’h6matocrite chez des individus diploldes et triploldes du Triton Pleurodeles waltlii. C. r. Seanc. Sot. Biol., Paris 160,416-419. FANKHAUSERG. (1945) The effect of changes in chromosome number on amphibian development. Q. Reu. Biol. 20, 20-78. JAYLET A. (1972) T6traploidie expCrimentale chez le Triton Pleurodeles waltlii Michah. Chromosoma 38, 173-184. KAWAMURAT. & NISHIOKAM. (1963) Autotetraploids and the production of allotetraploids and diploid nucleo-cytoplasmic hybrids in pond frogs. J. Sci. Hiroshima Univ. B 21, l-22. Key Word Index-Red blood cells; diploid; polyploid; hemoglobin; Pleurodeles.
5OA,pp. 263
to
266. Pergamon Press. Printed in Great Britain
RED BLOOD CELLS AND HEMOGLOBIN CONCENTRATION IN NORMAL DIPLOID AND SEVERAL TYPES OF POLYPLOID SALAMANDERS* PIERREDEPARIS,JEAN-CLAUDEBEETSCHEN ANDANDREJAYLET Laboratoire de Biologie g&&ale, Universite Paul-Sabatier, 118 route de Narbonne, 31077 ToulouseCedex, France (Received I November
1913)
Different blood parameters were examined in both diploid and polyploid adult Pieuro-
Abstract-l. deles newts. 2. In 2 n,
3 n and 4 n, the hemoglobin content and hematocrit values were very close. 3. The red blood cell counts decrease as the degree of polyploidy rises. 4. Autotetraploid and pentaploid animals do not regulate their hemoglobin values and are slightly
INTRODUCTION
diploid females (Beetschen, 1960), or by crossing female 4 n x male 2 n (Beetschen, 1962). Tetraploid animals were obtained either among the
A HOMEOSTATIC mechanism maintaining the concentration of hemoglobin for the species, in spite of triploidy and its effects on the erythrocyte size and number, has been recently described in adult intersex triploid chickens (Abdel-Hameed, 1972). Though not mentioned in that paper, such a homeostatic effect had already been studied in polyploids of the Urodele Amphibian, Pleurodeles waltlii, with respect to the number of erythrocytes (Deparis & Beetschen, 1965a), hematocrit and hemoglobin concentration (Deparis et al., 1965b). Although the red blood cell (RBC) number decreases in triploid and tetraploid salamanders, both triploid and diploid animals exhibit similar RBC packed volumes (hematocrit) and hemoglobin concentrations. We repeated such studies on several kinds of adult polyploid Pleurodeles now available in our laboratory (3 n, 4 n, 5 n), in order to check whether the regulation phenomena previously observed in triploid animals also occur in tetraploids and pentaploids. For this purpose we have in each case determined the RBC number, the concentration of hemoglobin and we have also measured the hematocrit value. With these results we have calculated the mean RBC volume, the mean corpuscular hemoglobin and the mean corpuscular hemoglobin concentration.
progeny of triploid females crossed with normal males (Beetschen, 1967), or by heating diploid eggs at the first furrow stage (autotetraploids) (Jaylet, 1972). Pentaploid animals were produced by cold treatment of the eggs of tetraploid females fertilized by normal sperm (Bee&hen, 1964). Erythrocyte counts were made with a Nageotte counting chamber, in 25 mm3 volume, after a l/400 part dilution of the blood in Marcano’s fluid. The amount of hemoglobin has been measured according to Drabkin’s method. The hematocrit value has been determined by the microhematocrit method. The following formulae were used in the calculations of: Mean Corpuscular Volume (M.C.V.) in ~3 = hematocrit % x 10 red cell count (millions/mm3)’ Mean Corpuscular Hemoglobin
(M.C.H.) in ppg =
hemoglobin (g/100 ml) red cell count (millions/mms)’ Mean
Corpuscular (M.C.H.C.)
Hemoglobin Concentration as a percentage =
hemoglobin (g/100 ml) volume of packed red cells (ml/l00 ml)’
MATERIALS AND METHODS
RESULTS
Triploid animals (Pleurodeles waltlii)were obtained either by cold treatment of fertilized eggs of normal * Supported by a CNRS grant in the field of developmental genetics in Urodele Amphibians (ERA No. 327).
The polyploidy causes a reduction in the number of RBC. Table 1 shows that the triploid erythrocytes are present in numbers which are about two-thirds of
263
264
PIERREDEPARIS,JEAN-CLAUDEBEETSCHENAND ANDR~JAYLET Table 1. Review table of the different erythrocyte constants in normal diploid individuals triploids (3 n), tetraploids (4 n), autotetraploids (4 n A) and pentaploids (5 n) of cases
R.B.C. (counts/mm3)
25 25 5 12 5
172,150~9400 122,100~5800 82,450+2100 65,800+ 5200 74,750 f 1500
NO.
Ploidy 2n 3n 4n 4nA 5n
Hematocrit 0
Hb content WOO ml)
R.B.C. volume (~3)
39.1+ 1 37.62 1 35*4&o+ 27.5 i 1 33.0* 1
10*510*2 le2rtO.4 9*7& I,7 7*1+@8 8~4+0*2
2290+ 87 30842 51 43002 250 4170t230 4418i406
(2 n),
Mean corpus- Mean corpuscular cular Hb Hb concentration 618rt24 84Ort 26 1172ri: 147 1078255 1122183
26.9 k 0.6 27.220.6 27.411.5 25-8 + 1.3 25.5& 26
& fiducial limit of the mean. amount of diploid erythrocytes, whereas tetraploid erythrocytes are in numbers which are about one-half the number of diploid erythrocytes, The amount of pentaploid RBC should be about 40 per cent of the mznber of diploid RBC, i.e. 70,000 mm3. The observed amount comes close to the theoretical number. Nevertheless, it must be pointed out that the amount of diploid RBC may vary within fairly broad limits (Deparis & Beetschen, 1965a) and theoretical values calculated from a series of animals are but an indication. In spite of this, it seems evident that the RBC from autotetraploid animals are present in amounts lower than they should be. A~totetraploid originate from several spawnings of normal diploid progenitors. The RBC number of the dipioid brothers of these autotetraploids is normal Consequently, anemia of the autotetraploids must be considered as a result of their peculiar genetic constitution. Regulation of the hematocrit and hemoglobin content is due to the increase in cell size. The mean RBC volume for triploid animals is about one-third greater than that of diploid animals. In tetraploid animals this RBG volume is about twice that of diploid animals. However, a greater variability is observed in the size of tetraploid cells as is shown by the fiducial limits of the mean. The results just shown with regard to the mean RBC volume are equally valid for the mean corpuscular hemoglobin. Finally, the mean corpuscular concentration in hemoglobin has been calculated for each ploidy. Because of its precision this constant is very interesting, It can be ascertained (Table 1) that the values obtained for the 2 n, 3 n and 4 n animals are very similar, thus showing that a regulating mechanism governs the hemoglobin concentration in 3 n and 4 n. However, autotetraploids and pentaploids are slightly anaemic. the
The histogram (Fig. 1) shows that the increase in ploidy is accompanied by a reciprocal decrease in the number of RBC, The increase in size of poly-
ploid cells counteracts the reduction in their number (Fankhauser, 1945). Therefore, the hematocrit value and hemoglobin concentration are identical for 2 n and 3 n. The slight difference between 2 IZand 3 n hematocrits is not significant for P = 0.05 (Student’s f-test for determining the difference between two averages). A regulation phenomenon still occurs in tetraploid animals, although the hematocrit value is significantly different from that of diploid animals tested in the present paper, but their hemoglobin concentration is almost the same and is very similar to that of the previously tested animals (Deparis & Beetschen, 1965a). Furthermore, the difference observed here is not si~ifi~nt for P=O*O5. The autotetraploid animals have a hematocrit and a hemoglobin content inferior to those of tetraploid animals obtained by crossing female 3 n x male 2 n. Thus, total regulation of the hemoglobin concentration does not occur in a certain type of tetraploidy and it would be interesting to find out if this same phenomenon exists in the anuran autotetraploids described by other authors (Kawamura & Nishioka, 1963). Such a comparative study should be most valuable, because the results from Bgak & Pueyo (1970) do not correspond with the same interpretation. These authors have compared diploid and natural tetraploid anurans from the genus ~dun~up~zry~us and indicated, for hemoglobin and cardiac LDH, a reduced activity or partial inactivation of genes in the tetraploid 0. americanus as compared to the diploid 0. cultripes. The ratio between the mean corpuscular hemoglobin values (MCH) of both species is: MCH americanus 4 nJMCH cultripes 2 n = l-11 ; for the mean corpuscular volume (MCV), the ratio is 4n/2 n = 1.19. The same calculation applied to our own results on PZeuradeies gives for MCH a ratio of 4 n/2 n = 1.91 and for MCV a ratio of 4 n/2 n = 1.87. These ratios are not those obtained from autotetraploid Pleurodeles for which they should be lower, but it is known that the populations of 0. americanus are polymorphic. We believe that the comparative study by Becak and Pueyo (1970) should have been done between diploid and tetraploid animals of the same species 0. americanus, of which a few diploid
Red blood cells and hemoglobin concentration in salamanders
265
I80 170 160 150 140 130
30-
I20 25woo ; 90 z
_ 80 70
d
60
G
50
$
40
E 30 z" 20 mg
IO ; 110:
Fig. 1. Different erythrocyte constants in diploid and polyploid individuals. The vertical lines represent the fiducial limit of the mean. Diploid individuals (2 n), triploids (3 n), tetraploids (4 n), autotetraploids (4 n A) and pentaploids (5 n). populations are known, and not between two different species, before a firm conclusion could be given on this problem of gene regulation. Of course, the situation may be different in anuran and urodelan species. In pentaploid Pleurodeles the regulation of hematocrit and hemoglobin content no longer takes place. It is not a question of size or weight as these were shown to be normal but, as is shown in Table 1, it is due to the mean RBC volume which is lower than it should be according to theoretical calculations. The volume of a pentaploid erythrocyte of Pleurodeles should be 5500 ~3 but in fact it is only 4418 ~3. Here also there is an important variability in size. The hemoglobin content per cell is also lower than it should be, theoretically, and is a
little different from that of the tetraploid cells. However, the size of pentaploid cells is different from that of the tetraploid ones. The calculated values show a certain heterogeneity in the size of Figure 1 the pentaploid cellular population. indicates only the average numbers. Thus, a perfect hemeostatic mechanism would seem to be incompatible with a high degree of polyploidy as shown by pentaploid animals, but still occurs in at least one category of tetraploids. REFERENCES F. (1972) Hemoglobin concentration in normal diploid and intersex triploid chickens: genetic inactivation or canalization? Science, Wash. 178, 864-865.
ABDEL-HAMEED
266
Pierre DEPARIS,JEAN-CLAUDE BEERWHENAND ANDRI’ZJAYLET
BECAKW. & PUEYOM. T. (1970) Gene regulation in the polyploid Amphibian Odontophrynus americanus. Expl cell Res. 63, 448450. BEETSCHENJ. C. (1960) Recherches sur I’hCtCroploidie expkrimentale chez un Amphibien Urodkle, PIeurodeles waltlii Michah. BUN. bioI. France Belg. 94, 12-127. BEETSCHEN J. C. (1962) Sur la descendance de femelles t&raploides croistes avec des m&les diploldes chez l’amphibien urodele Pleurodeles waltlii. C. r. hebd. Sganc. Acad. Sci., Paris 255, 3068-3070. BEETSCHEN J. C. (1964) Pentaplofdie expkrimentale chez l’amphibien urodkle Pleurodeles waltlii Michah. C. r. hebd. Siam. Acad. Sci., Paris, 258, 1641-1643. BEE~SCHENJ. C. (1967) Cinq g&&rations d’individus polyploides chez le Triton Pleurodeles waltlii Michah. C. r. Skanc. Sot. Biol., Paris 161, 93G936. DEPARIS P. & BEETSCHENJ. C. (1965a) RBsultats comparatifs de numerations globulaires faites sur le sang d’individus diploldes et polyplofdes du Triton Pleuro-
deles waltlii Michah. C. r. SPanc. Sot. Biol., Paris 159, 12241229. DEPARIS P., NOUVEL H. & BEETSCHENJ. C. (1965b) Taux d’h6moglobine et valeur de l’h6matocrite chez des individus diploldes et triploldes du Triton Pleurodeles waltlii. C. r. Seanc. Sot. Biol., Paris 160,416-419. FANKHAUSERG. (1945) The effect of changes in chromosome number on amphibian development. Q. Reu. Biol. 20, 20-78. JAYLET A. (1972) T6traploidie expCrimentale chez le Triton Pleurodeles waltlii Michah. Chromosoma 38, 173-184. KAWAMURAT. & NISHIOKAM. (1963) Autotetraploids and the production of allotetraploids and diploid nucleo-cytoplasmic hybrids in pond frogs. J. Sci. Hiroshima Univ. B 21, l-22. Key Word Index-Red blood cells; diploid; polyploid; hemoglobin; Pleurodeles.
