Departments of Obstetrics and Gynaecology1) Physiological Chemistry2), University of Umeâ,
and and
Department
S-901 87 Umeâ, Sweden of Medicine I, Royal Vet. College3), Stockholm
DEMONSTRATION OF ANALOGUES TO THE HUMAN PREGNANCY ZONE PROTEIN IN ANIMALS
By Bo
Schoultz Martinsson
von
Kjell
Torgny Stigbrand Holmgren
and Nils
ABSTRACT The pregnancy zone protein (PZ) is known to occur in increased amounts during pregnancy and hormonal treatment. Analogues of this human serum \g=a\2-globulinwere demonstrated in animals by double gel diffusion and immunoelectrophoresis. Direct cross-reactions were found for Rhesus monkey and beagle dog against anti-human pregnancy zone protein. After preparation of an "anti-dog PZ" serum it was possible to demonstrate analogues also in cow and sheep. Thus, the present investigation demonstrates animal analogues to the human pregnancy zone protein, which should be valuable for studies of the biological role of this protein.
Since the first report of the so called pregnancy zone protein (Smithies 1959), there have been several studies on this human o^-serum globulin (Afonso 8c Farnham 1962; Afonso 8c de Alvarez 1963, 1964; Cooper 1963; de Alvarez 8c Thompson 1964; Robinson et al. 1966; de Alvarez 8c Afonso 1967; Margolis 8c Kenrick 1969). This protein (PZ) is found only in low concentrations in nonpregnant women and men but is during pregnancy induced an increased amounts averaging 100 mg/100 ml during the last half of pregnancy (von Schoultz 1974; Damber et al. 1975, in press). PZ has been purified (von Schoultz 8c Stigbrand 1973) and characterized (von Schoultz 8c Stigbrand 1974). The purified PZ-protein has been found to
inhibit the PHA-induced stimulation of lymphocytes, and PZ might have immunosuppressive properties (von Schoultz et al. 1974). When discussing the biological role of PZ it is important to know whether the induction of this high molecular weight protein in highly increased amounts is a phenomenon peculiar to human pregnancy or if the same process can be observed in pregnant animals. Hofmann et al. (1972) were unable to demon¬ strate the pregnancy zone protein in the sera of rabbits, mice, guinea pigs or rats. Bohn 8c Ronneberger (1973) could demonstrate an analogue to SP3 in the serum from a pregnant Rhesus monkey. They found no corresponding proteins in dog or in several small rodents as rat, rabbit, mouse, hamster and guinea pig. The present investigation describes the presence of animal counterparts to the human pregnancy zone protein.
MATERIAL AND METHODS Serum samples were taken from two pregnant beagle dogs, swine, sera. sheep, horses and Rhesus monkeys, respectively. The sera were collected between 2-7 days before delivery, and after centrifugation stored at -20°C until use. Antiserum against human PZ. The purified PZ-protein (von Schoultz Se Stigbrand 1973) was injected into rabbits and a monospecific precipitating antiserum was pre¬ pared as described in a previous publication (Beckman et al. 1973). lmmunological methods. Double diffusion experiments were performed according to Ouchtcrlony (1948) using 1 °/o agarose slides (Miles, Co.) and immunoelectrophoresis was performed in 1 */o agarose in 0.024 M veronal buffer pH 8.6 according to Scheideggar (1955). Antisera against animal PZ. Double diffusion precipitates from pregnant beagle dog sera cross-reacting with antihuman PZ were used for immunization of rabbits as described by Martinsson (1974) and Holmgren (1973). The precipitates were carefully cut out, washed, and then frozen at -20°C until use. The suspension formed after thawing was injected into rabbits. Each dose for immunization contained 10-12 "rings" of precipitates (each ring consisted of 6 precipitates). Gel chromatography. Two ml serum from a pregnant dog was applied on a Sephadex G-200 Superfine column (2.5x50 cm) in 0.15 M NaCl buffered with 20 mM sodium phopshate pH 7.4. Protein was recorded by UV absorption at 280 nm. Fractions were tested for the presence of the PZ-analogue using the prepared "anti dog-PZ" Animal
—
cows,
-
—
-
-
serum.
RESULTS AND DISCUSSION
The antihuman PZ-serum was found to precipitate sera from pregnant beagle dogs and Rhesus monkeys in both immunoelectrophoretic (Fig. 1) and double gel diffusion (Fig. 2) experiments. In both experiments only one precipitate could be observed. In immunoelectrophoresis these precipitates had about the
Fig. 1. Immunoelectrophoresis using anti-human "pregnancy zone" protein against sera from (1) Rhesus monkey, (2) man) and (3) dog.
Fig.
pregnancy
2.
Double-diffusion experiment, demonstrating analogues to human PZ. Anti-human pregnancy zone protein in the central well. In the peripheral wells pregnancy sera from (1) Rhesus monkey, (2) man, (3) dog, (4) and (5) horse.
Fig. 3. Immunoelectrophoresis using anti-dog "pregnancy zone" protein against from (1) sheep, (2) dog and (3) cow.
Fig.
pregnancy
sera
4.
Double-diffusion experiment using anti-dog "pregnancy zone" protein the the central. well. In the peripheral wells pregnancy sera from (1) sheep, (2) dog, (3) cow and (4) horse. Note the apparent identity between cow and sheep and the partial identity between these sera and the dog sera.
Fig. 5. using (1) anti-human pregnancy zone protein, zone protein and (3) pregnant dog serum.
Immuno-diffusion experimens
(2) anti-dog
pregnancy
position as the precipitate of human PZ (Fig. 1). Sera from pregnant cows, sheep and horses gave no visible cross-reactions. The precipitates formed by pregnant dog serum against anti-human PZ-
same
swine,
used to prepare a rabbit "anti-dog-PZ" serum. The most potent rabbit antiserum was used. This "anti-dog-PZ" serum was found to give one single precipitate with pregnant dog serum in both immunoelectrophoresis (Fig. 3) and double gel diffusion (Fig. 4). The position of the precipitate in immunoelectrophoresis was in the a-region. This new 'anti-dog PZ" serum in turn was found to strongly cross-react with sera from pregnant cow and sheep (Figs. 3 and 4), and only one precipitate could be seen. Furthermore double diffusion tests revealed a partial identity between the PZ-analogue in dog and cow and dog and sheep, respectively (Fig. 4). The protein from cow and sheep seems to give a complete fusion reaction, sera from pregnant horse and swine revealed no or very faint precipitates with "anti-dog-PZ" serum. In Fig. 5 dog serum is shown to react with both anti-human PZ serum and "antidog-PZ" serum. By gel filtration of pregnant dog serum the PZ-analogue was identified in the macroglobulin peak. Preliminary studies in non-pregnant animals (cows and dogs) indicate a relatively high concentration of PZserum were
analogue (Martinsson et al., unpublished observation). It seems likely that an extended procedure and preparation of various spécifique "anti-animal-PZ" sera should reveal analogues also in several other species. To elucidate the biological role of PZ animal models should be most valuable. The preparation of an antiserum against this protein in some ex¬ perimental animals should facilitate such studies. Furthermore there may be a selective veterinary interest in investigations on the presence of the pregnancy zone protein in some animals regarding pregnancy diagnosis.
ACKNOWLEDGMENTS This work was financially supported by grants to T. S. and B. v. S. from the Swedish Medical Research Council (Project 4217) and from the "Tore Nilssons fond for Medical Research". Skilful technical assistance was provided by Mrs. K. Hjortsberg, Mrs. M. Isaksson and Mrs. M. Wallen.
REFERENCES
Afonso J. F. Afonso J. F. Afonso J. F.
Se de Alvarez R. R.: Amer. J. Obstet. Gynec. 86 (1963) 815. Se de Alvarez R. R.: Amer. J. Obstet. Gynec. 89 (1964) 204. Se Farnham N. G.: Amer. J. Obstet. Gynec. 84 (1962) 199. de Alvarez R. R. Se Afonso J. F.: Penn. med. J. 70 (1967) 43. de Alvarez R. R. Se Thompson I. E.: Obstet, and Gynec. 23 (1964) 640. Beckman L.. von Schultz B. Se Stigbrand T.: Acta, obstet, gynec. scand. 52 (1973) 157. Bohn H. Se Ronneberger H.: Arch. Gynäk. 215 (1973) 277. Cooper D. W.: Nature (Lond.) 200 (1963) 892. Hofmann R., Straube W., Klausch B., Friemel H. Se Günther J.: Arch. Gynäk. 212 (1972) 246. Holmgren N'.: Acta vet. scand. 14 (1973) 366. Margolis ]. Se Kenrick K. G.: Aust. J. exp. Biol. med. Sei. 47 (1969) 637. Martinsson K.: Zbl. Vet.-Med. B 21 (1974) 302. Ouchterlony O.: Acta path, microbiol. scand. 25 (1948) 186. Robinson J. C, London W. T. Se Pierce J. E.: Amer. J. Obstet. Gynec. 96 (1966) 226. Scheideggar ].: Int. Archs Allergy appl. Immunol. 7 (1955) 103. von Schoultz B.: Amer. J. Obstet. Gynec. 119 (1974) 792. von Schoultz B. Se Stigbrand T.: Acta obstet, gynec. scand. 52 (1973) 51. von Schoultz B. Se Stigbrand T.: Biochim. biophys. Acta (Amst.) 359 (1974) 303. von Schoultz B., Stigbrand T. Se Tärnvik A.: FEBS Letters 38 (1974) 23. Smithies O.: Advanc. Protein Chem. 14 (1959) 65.
Received
on
February 4th,
1975.
and and
Department
S-901 87 Umeâ, Sweden of Medicine I, Royal Vet. College3), Stockholm
DEMONSTRATION OF ANALOGUES TO THE HUMAN PREGNANCY ZONE PROTEIN IN ANIMALS
By Bo
Schoultz Martinsson
von
Kjell
Torgny Stigbrand Holmgren
and Nils
ABSTRACT The pregnancy zone protein (PZ) is known to occur in increased amounts during pregnancy and hormonal treatment. Analogues of this human serum \g=a\2-globulinwere demonstrated in animals by double gel diffusion and immunoelectrophoresis. Direct cross-reactions were found for Rhesus monkey and beagle dog against anti-human pregnancy zone protein. After preparation of an "anti-dog PZ" serum it was possible to demonstrate analogues also in cow and sheep. Thus, the present investigation demonstrates animal analogues to the human pregnancy zone protein, which should be valuable for studies of the biological role of this protein.
Since the first report of the so called pregnancy zone protein (Smithies 1959), there have been several studies on this human o^-serum globulin (Afonso 8c Farnham 1962; Afonso 8c de Alvarez 1963, 1964; Cooper 1963; de Alvarez 8c Thompson 1964; Robinson et al. 1966; de Alvarez 8c Afonso 1967; Margolis 8c Kenrick 1969). This protein (PZ) is found only in low concentrations in nonpregnant women and men but is during pregnancy induced an increased amounts averaging 100 mg/100 ml during the last half of pregnancy (von Schoultz 1974; Damber et al. 1975, in press). PZ has been purified (von Schoultz 8c Stigbrand 1973) and characterized (von Schoultz 8c Stigbrand 1974). The purified PZ-protein has been found to
inhibit the PHA-induced stimulation of lymphocytes, and PZ might have immunosuppressive properties (von Schoultz et al. 1974). When discussing the biological role of PZ it is important to know whether the induction of this high molecular weight protein in highly increased amounts is a phenomenon peculiar to human pregnancy or if the same process can be observed in pregnant animals. Hofmann et al. (1972) were unable to demon¬ strate the pregnancy zone protein in the sera of rabbits, mice, guinea pigs or rats. Bohn 8c Ronneberger (1973) could demonstrate an analogue to SP3 in the serum from a pregnant Rhesus monkey. They found no corresponding proteins in dog or in several small rodents as rat, rabbit, mouse, hamster and guinea pig. The present investigation describes the presence of animal counterparts to the human pregnancy zone protein.
MATERIAL AND METHODS Serum samples were taken from two pregnant beagle dogs, swine, sera. sheep, horses and Rhesus monkeys, respectively. The sera were collected between 2-7 days before delivery, and after centrifugation stored at -20°C until use. Antiserum against human PZ. The purified PZ-protein (von Schoultz Se Stigbrand 1973) was injected into rabbits and a monospecific precipitating antiserum was pre¬ pared as described in a previous publication (Beckman et al. 1973). lmmunological methods. Double diffusion experiments were performed according to Ouchtcrlony (1948) using 1 °/o agarose slides (Miles, Co.) and immunoelectrophoresis was performed in 1 */o agarose in 0.024 M veronal buffer pH 8.6 according to Scheideggar (1955). Antisera against animal PZ. Double diffusion precipitates from pregnant beagle dog sera cross-reacting with antihuman PZ were used for immunization of rabbits as described by Martinsson (1974) and Holmgren (1973). The precipitates were carefully cut out, washed, and then frozen at -20°C until use. The suspension formed after thawing was injected into rabbits. Each dose for immunization contained 10-12 "rings" of precipitates (each ring consisted of 6 precipitates). Gel chromatography. Two ml serum from a pregnant dog was applied on a Sephadex G-200 Superfine column (2.5x50 cm) in 0.15 M NaCl buffered with 20 mM sodium phopshate pH 7.4. Protein was recorded by UV absorption at 280 nm. Fractions were tested for the presence of the PZ-analogue using the prepared "anti dog-PZ" Animal
—
cows,
-
—
-
-
serum.
RESULTS AND DISCUSSION
The antihuman PZ-serum was found to precipitate sera from pregnant beagle dogs and Rhesus monkeys in both immunoelectrophoretic (Fig. 1) and double gel diffusion (Fig. 2) experiments. In both experiments only one precipitate could be observed. In immunoelectrophoresis these precipitates had about the
Fig. 1. Immunoelectrophoresis using anti-human "pregnancy zone" protein against sera from (1) Rhesus monkey, (2) man) and (3) dog.
Fig.
pregnancy
2.
Double-diffusion experiment, demonstrating analogues to human PZ. Anti-human pregnancy zone protein in the central well. In the peripheral wells pregnancy sera from (1) Rhesus monkey, (2) man, (3) dog, (4) and (5) horse.
Fig. 3. Immunoelectrophoresis using anti-dog "pregnancy zone" protein against from (1) sheep, (2) dog and (3) cow.
Fig.
pregnancy
sera
4.
Double-diffusion experiment using anti-dog "pregnancy zone" protein the the central. well. In the peripheral wells pregnancy sera from (1) sheep, (2) dog, (3) cow and (4) horse. Note the apparent identity between cow and sheep and the partial identity between these sera and the dog sera.
Fig. 5. using (1) anti-human pregnancy zone protein, zone protein and (3) pregnant dog serum.
Immuno-diffusion experimens
(2) anti-dog
pregnancy
position as the precipitate of human PZ (Fig. 1). Sera from pregnant cows, sheep and horses gave no visible cross-reactions. The precipitates formed by pregnant dog serum against anti-human PZ-
same
swine,
used to prepare a rabbit "anti-dog-PZ" serum. The most potent rabbit antiserum was used. This "anti-dog-PZ" serum was found to give one single precipitate with pregnant dog serum in both immunoelectrophoresis (Fig. 3) and double gel diffusion (Fig. 4). The position of the precipitate in immunoelectrophoresis was in the a-region. This new 'anti-dog PZ" serum in turn was found to strongly cross-react with sera from pregnant cow and sheep (Figs. 3 and 4), and only one precipitate could be seen. Furthermore double diffusion tests revealed a partial identity between the PZ-analogue in dog and cow and dog and sheep, respectively (Fig. 4). The protein from cow and sheep seems to give a complete fusion reaction, sera from pregnant horse and swine revealed no or very faint precipitates with "anti-dog-PZ" serum. In Fig. 5 dog serum is shown to react with both anti-human PZ serum and "antidog-PZ" serum. By gel filtration of pregnant dog serum the PZ-analogue was identified in the macroglobulin peak. Preliminary studies in non-pregnant animals (cows and dogs) indicate a relatively high concentration of PZserum were
analogue (Martinsson et al., unpublished observation). It seems likely that an extended procedure and preparation of various spécifique "anti-animal-PZ" sera should reveal analogues also in several other species. To elucidate the biological role of PZ animal models should be most valuable. The preparation of an antiserum against this protein in some ex¬ perimental animals should facilitate such studies. Furthermore there may be a selective veterinary interest in investigations on the presence of the pregnancy zone protein in some animals regarding pregnancy diagnosis.
ACKNOWLEDGMENTS This work was financially supported by grants to T. S. and B. v. S. from the Swedish Medical Research Council (Project 4217) and from the "Tore Nilssons fond for Medical Research". Skilful technical assistance was provided by Mrs. K. Hjortsberg, Mrs. M. Isaksson and Mrs. M. Wallen.
REFERENCES
Afonso J. F. Afonso J. F. Afonso J. F.
Se de Alvarez R. R.: Amer. J. Obstet. Gynec. 86 (1963) 815. Se de Alvarez R. R.: Amer. J. Obstet. Gynec. 89 (1964) 204. Se Farnham N. G.: Amer. J. Obstet. Gynec. 84 (1962) 199. de Alvarez R. R. Se Afonso J. F.: Penn. med. J. 70 (1967) 43. de Alvarez R. R. Se Thompson I. E.: Obstet, and Gynec. 23 (1964) 640. Beckman L.. von Schultz B. Se Stigbrand T.: Acta, obstet, gynec. scand. 52 (1973) 157. Bohn H. Se Ronneberger H.: Arch. Gynäk. 215 (1973) 277. Cooper D. W.: Nature (Lond.) 200 (1963) 892. Hofmann R., Straube W., Klausch B., Friemel H. Se Günther J.: Arch. Gynäk. 212 (1972) 246. Holmgren N'.: Acta vet. scand. 14 (1973) 366. Margolis ]. Se Kenrick K. G.: Aust. J. exp. Biol. med. Sei. 47 (1969) 637. Martinsson K.: Zbl. Vet.-Med. B 21 (1974) 302. Ouchterlony O.: Acta path, microbiol. scand. 25 (1948) 186. Robinson J. C, London W. T. Se Pierce J. E.: Amer. J. Obstet. Gynec. 96 (1966) 226. Scheideggar ].: Int. Archs Allergy appl. Immunol. 7 (1955) 103. von Schoultz B.: Amer. J. Obstet. Gynec. 119 (1974) 792. von Schoultz B. Se Stigbrand T.: Acta obstet, gynec. scand. 52 (1973) 51. von Schoultz B. Se Stigbrand T.: Biochim. biophys. Acta (Amst.) 359 (1974) 303. von Schoultz B., Stigbrand T. Se Tärnvik A.: FEBS Letters 38 (1974) 23. Smithies O.: Advanc. Protein Chem. 14 (1959) 65.
Received
on
February 4th,
1975.
