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Blastokinin In The Northern Fur Seal*
Vol. 23, No. 2, February 1972 Printed in U.S.A.
fERTILITY AND STERILITY
Copyright © 1972 by The Williams & Wilkins Co.
BLASTOKININ IN THE NORTHERN FUR SEAL* JOSEPH C. DANIEL, JR. PH.D.t
Department of Molecular, Cellular, and Developmental Biology, University of Colorqdo, Boulder, Colorado
The diapausing blastocysts of a variety of mammals having delayed implantation become activated when cultured in a medium supplemented with blastokinin (BKN) from the rabbit. 1 However, using Sephadex gel filtration and/or disc acrylamide electrophoresis, it has been impossible to demonstrate, convincingly and repeatedly, whether the uterine secretions of these mammals contain a blastokininlike component. 1 - 3 The more sensitive methods of immunoassay and pulse-power slab acrylamide electrophoresis (Ortec Inc., Oak Ridge, Tenn.) have now been used to test whether such a component exists in the uterine fluids of the Northern Fur Seal. METHODS AND MATERIALS
Fur seal uteri were obtained by the author in August, 1970 from animals killed in the annual fur harvest on the Pribilof Islands. The uteri were flushed with buffered saline solution and the flushings pooled from 10 animals were frozen for shipment back to the laboratory in Colorado. There the flushings were thawed, centrifuged to remove cellular debris, dialyzed for 24 hr. against distilled water, and then lyophilized to a dry powder. This powder was used as the sample for protein analysis. The Ortec system for pulse- power slab acrylamide gel electrophoresis has been described in detail elsewhere. 4 For Received August 5, 1971. • Supported by NIH Grants R01 HD 04165 and HD02282. t Present address: Department of Zoology and Entomology, University of Tennessee, Knoxville, Tenn.
these studies I used 4 1/:l% well-forming gel layer with 12 wells. Each sample was applied as 50 ml. of a solution made by dissolving 5 mg. of the sample powder in 1 ml. of citrate buffer and 50% sucrose overlaying with an 8% gel layer. For comparison rabbit uterine fluids, 'Collected on Day 5 post coitum were always run along with the fur seal samples. The immunoassay used was that developed by Johnson, Cowan, and Daniel, 5 reported in this issue. Radial immunodiffusion plates were prepared using absorbed antiserum from goats. The sample powder obtained from lyophilization of dialyzed uterine flushings was dissolved in buffered saline solution at a concentration of 5 mg./ml. This solution was used full strength and in dilutions of 1/2, V4, 1/s, Yl6, YJ2, and \164. A second sample was prepared in an attempt to concentrate any possible blastokinin component. Uterine flushings were fractionated by filtration on Sephadex gel G-200. The fractions that corresponded to those where blastokinin commonly occurs after filtration of rabbit uterine fluids were again dialyzed and lyophilized and this powder tested in the same way as the first, unfractionated sample. Blastokinin from the rabbit, purified in the same way was used for reference in each plate. Diffusion was permitted to proceed for 72 hr., observations being made every 12 hr. The plates were then stained with Amido black and precipitation bands noted. RESULTS
Figure 1 is the pattern obtained from electrophoresis, showing the comparison
78
February 1972
79
BLASTOKININ
FIG. 1. Electrophoresis of uterine fluids of the rabbit (R) and fur seal (F). The albumin (A) and blastokinin (B) bands are indicated.
between rabbit and fur seal uterine fluids. Obviously, both contain bands at the usual locus for albumin and blastokinin and possibly some other less well-defined components. The difference in the mobility of albumin may be a species difference or it may reflect the observation of Cowan and Daniel 6 regarding changes in uterine fluid albumin. In the immunoassays, neither the whole uterine fluid sample nor the isolated component sample caused any precipitation bands to develop that indicated a reaction with anti-BKN serum.
amount of protein present in uterine fluids during delayed implantation. It may be noted, however, that there is some evidence of a component having the same molecular weight as blastokinin found in Sephadex gel filtration studies of fur seal uterine fluids taken near the time of implantation. 3 The negative results from the immunoassay seem to support the conclusion that the protein from rabbits differs structurally from that of the fur seal. It could however reflect again the problem of low concentration, bound molecules that cover or alter reaction sites, or of denaturation by the isolation procedures. Even in the electrophoresis studies it is obvious that, in the fur seal the concentration of the blastokinin-like component is very low, as are all of the uterine proteins, during the period of embryonic diapause. But, the existence of a unique substance in two such unrelated species of mammals would seem to argue for a common need and implicate the substance in some fundamental process. SUMMARY
Electrophoresis of fur seal uterine fluids collected during the delay preceeding implantation, shows a band that migrates like blastokinin in the rabbit. Total protein levels are much lower in the fur seal and the BKN-like component composes a much smaller fraction of the proteins than is the case in the rabbit. Apparently the BKN component differs qualitatively between the two speDISCUSSION cies. Precipitation bands that form when From these observations, it is seen rabbit BKN is tested by radial immunothat a proteinaceous component may be diffusion against goat anti-BKN serum found in the uterine secretions of the do not form when fur seal uterine fluids Northern Fur Seal that has electro- or the isolated components are used. phoretic properties similar to those of On the basis of molecular weight and blastokinin from the rabbit. Presumably electrophoretic mobility, one concludes the difficulty of identifying this com- that a component similar to blastokinin ponent in earlier studies was related to exists in the uterine fluids of the Northern the problems of fractionating the small Fur Seal. This component differs in im-
80
DANIEL
munologic properties from that isolated from the rabbit uterus. Acknowledgment. The author is grateful to Mrs. Susan MacFadden for technical assistance.
3.
4.
REFERENCES 1. DANIEL, J. C., AND KRISHNAN, R. S. Studies on the relationship between uterine fluid components and the diapausing state of blastocysts from mammals having delayed implantation. J Exp Zool 172:267, 1969. 2. DANIEL, J. C. Comparison of electrophoretic patterns of uterine fluids from rabbits and mam-
5.
6.
Vol. 23 mals having delayed implantation. Camp Biochem Physiol 24:297, 1968. DANIEL, J. C. Growth of the preimplantation embryo of the northern fur seal and its correlation with changes in uterine protein. Develop Biol 26:316, 1971. MuRRAY, F. A., McGAuGHEY, R. W., AND YARus, M. J. Blastokinin: Its size and shape, and an indication of the existence of subunits. Fertil Steril 23:69, 1972. JoHNSON, M. H., CowAN, B. D., AND DANIEL, J. C. An immunological assay for blastokinin. Fertil Steril 23:93, 1972. CowAN, B. D., AND DANIEL, J. C. Difference in electrophoretic mobility between the albumin of rabbit uterine fluid and serum albumin. Fertil Steril 23:81, 1972.
fERTILITY AND STERILITY
Copyright © 1972 by The Williams & Wilkins Co.
BLASTOKININ IN THE NORTHERN FUR SEAL* JOSEPH C. DANIEL, JR. PH.D.t
Department of Molecular, Cellular, and Developmental Biology, University of Colorqdo, Boulder, Colorado
The diapausing blastocysts of a variety of mammals having delayed implantation become activated when cultured in a medium supplemented with blastokinin (BKN) from the rabbit. 1 However, using Sephadex gel filtration and/or disc acrylamide electrophoresis, it has been impossible to demonstrate, convincingly and repeatedly, whether the uterine secretions of these mammals contain a blastokininlike component. 1 - 3 The more sensitive methods of immunoassay and pulse-power slab acrylamide electrophoresis (Ortec Inc., Oak Ridge, Tenn.) have now been used to test whether such a component exists in the uterine fluids of the Northern Fur Seal. METHODS AND MATERIALS
Fur seal uteri were obtained by the author in August, 1970 from animals killed in the annual fur harvest on the Pribilof Islands. The uteri were flushed with buffered saline solution and the flushings pooled from 10 animals were frozen for shipment back to the laboratory in Colorado. There the flushings were thawed, centrifuged to remove cellular debris, dialyzed for 24 hr. against distilled water, and then lyophilized to a dry powder. This powder was used as the sample for protein analysis. The Ortec system for pulse- power slab acrylamide gel electrophoresis has been described in detail elsewhere. 4 For Received August 5, 1971. • Supported by NIH Grants R01 HD 04165 and HD02282. t Present address: Department of Zoology and Entomology, University of Tennessee, Knoxville, Tenn.
these studies I used 4 1/:l% well-forming gel layer with 12 wells. Each sample was applied as 50 ml. of a solution made by dissolving 5 mg. of the sample powder in 1 ml. of citrate buffer and 50% sucrose overlaying with an 8% gel layer. For comparison rabbit uterine fluids, 'Collected on Day 5 post coitum were always run along with the fur seal samples. The immunoassay used was that developed by Johnson, Cowan, and Daniel, 5 reported in this issue. Radial immunodiffusion plates were prepared using absorbed antiserum from goats. The sample powder obtained from lyophilization of dialyzed uterine flushings was dissolved in buffered saline solution at a concentration of 5 mg./ml. This solution was used full strength and in dilutions of 1/2, V4, 1/s, Yl6, YJ2, and \164. A second sample was prepared in an attempt to concentrate any possible blastokinin component. Uterine flushings were fractionated by filtration on Sephadex gel G-200. The fractions that corresponded to those where blastokinin commonly occurs after filtration of rabbit uterine fluids were again dialyzed and lyophilized and this powder tested in the same way as the first, unfractionated sample. Blastokinin from the rabbit, purified in the same way was used for reference in each plate. Diffusion was permitted to proceed for 72 hr., observations being made every 12 hr. The plates were then stained with Amido black and precipitation bands noted. RESULTS
Figure 1 is the pattern obtained from electrophoresis, showing the comparison
78
February 1972
79
BLASTOKININ
FIG. 1. Electrophoresis of uterine fluids of the rabbit (R) and fur seal (F). The albumin (A) and blastokinin (B) bands are indicated.
between rabbit and fur seal uterine fluids. Obviously, both contain bands at the usual locus for albumin and blastokinin and possibly some other less well-defined components. The difference in the mobility of albumin may be a species difference or it may reflect the observation of Cowan and Daniel 6 regarding changes in uterine fluid albumin. In the immunoassays, neither the whole uterine fluid sample nor the isolated component sample caused any precipitation bands to develop that indicated a reaction with anti-BKN serum.
amount of protein present in uterine fluids during delayed implantation. It may be noted, however, that there is some evidence of a component having the same molecular weight as blastokinin found in Sephadex gel filtration studies of fur seal uterine fluids taken near the time of implantation. 3 The negative results from the immunoassay seem to support the conclusion that the protein from rabbits differs structurally from that of the fur seal. It could however reflect again the problem of low concentration, bound molecules that cover or alter reaction sites, or of denaturation by the isolation procedures. Even in the electrophoresis studies it is obvious that, in the fur seal the concentration of the blastokinin-like component is very low, as are all of the uterine proteins, during the period of embryonic diapause. But, the existence of a unique substance in two such unrelated species of mammals would seem to argue for a common need and implicate the substance in some fundamental process. SUMMARY
Electrophoresis of fur seal uterine fluids collected during the delay preceeding implantation, shows a band that migrates like blastokinin in the rabbit. Total protein levels are much lower in the fur seal and the BKN-like component composes a much smaller fraction of the proteins than is the case in the rabbit. Apparently the BKN component differs qualitatively between the two speDISCUSSION cies. Precipitation bands that form when From these observations, it is seen rabbit BKN is tested by radial immunothat a proteinaceous component may be diffusion against goat anti-BKN serum found in the uterine secretions of the do not form when fur seal uterine fluids Northern Fur Seal that has electro- or the isolated components are used. phoretic properties similar to those of On the basis of molecular weight and blastokinin from the rabbit. Presumably electrophoretic mobility, one concludes the difficulty of identifying this com- that a component similar to blastokinin ponent in earlier studies was related to exists in the uterine fluids of the Northern the problems of fractionating the small Fur Seal. This component differs in im-
80
DANIEL
munologic properties from that isolated from the rabbit uterus. Acknowledgment. The author is grateful to Mrs. Susan MacFadden for technical assistance.
3.
4.
REFERENCES 1. DANIEL, J. C., AND KRISHNAN, R. S. Studies on the relationship between uterine fluid components and the diapausing state of blastocysts from mammals having delayed implantation. J Exp Zool 172:267, 1969. 2. DANIEL, J. C. Comparison of electrophoretic patterns of uterine fluids from rabbits and mam-
5.
6.
Vol. 23 mals having delayed implantation. Camp Biochem Physiol 24:297, 1968. DANIEL, J. C. Growth of the preimplantation embryo of the northern fur seal and its correlation with changes in uterine protein. Develop Biol 26:316, 1971. MuRRAY, F. A., McGAuGHEY, R. W., AND YARus, M. J. Blastokinin: Its size and shape, and an indication of the existence of subunits. Fertil Steril 23:69, 1972. JoHNSON, M. H., CowAN, B. D., AND DANIEL, J. C. An immunological assay for blastokinin. Fertil Steril 23:93, 1972. CowAN, B. D., AND DANIEL, J. C. Difference in electrophoretic mobility between the albumin of rabbit uterine fluid and serum albumin. Fertil Steril 23:81, 1972.