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The Effect of Protein Fractions from Rabbit Uterine Fluids on Embryo Growth and Uptake of Nucleic Acid and Protein Precursors*
VoL 23, No.2, February 1972 Printed in U.S.A.
FERTILITY AND STERILITY
Copyright © 1972 by The Williams & Wilkins Co.
THE EFFECT OF PROTEIN FRACTIONS FROM RABBIT UTERINE FLUIDS ON EMBRYO GROWTH AND UPTAKE OF NUCLEIC ACID AND PROTEIN PRECURSORS* AMIN A. EL-BANNA, PH.D., t
AND
JOSEPH C. DANIEL, JR., PH.D.:j:
Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, Colorado
Specific proteins appear in the uterine secretions of mammals during pregnancy and at times that coincide with specific developmental stages of their embryos. Such coincidence has prompted investigators to assume that dependent interrelationships exist: primarily that one or more of the proteins are essential for growth and/or differentiation of the corresponding stage embryo. 1 - 3 One especially prominent protein is secreted by the uterus of the rabbit during the period when the embryos are free-living, unattached blastocysts. When tested in vitro, this protein, "blastokinin," stimulated blastulation of morulae, 1 growth of blastocysts, 3 new mitotic activity in "dormant" blastocysts, • and synthesis of protein 5 and ribonucleic acid (RNA) 6 as measured by uptake of radiolabeled precursors. To the best of our knowledge this is the only uterine protein that has been tested for its effects on embryos even though as many as 18 separate proteins (some of serum origin) have been reported to exist in the rabbit uterus. By G-200 Sephadex gel filtration of rabbit uterine proteins collected in early pregnancy, prior to implantation, six different molecular weight
fractions can be identified and isolated: The present report concerns the results of studies designed to test the comparative effects of each of these six fractions on rabbit morulae and blastocysts in vitro. METHODS AND MATERIALS
Uterine fluids were collected from rabbits on the 3rd and 5th day postcoitum. Each isolated uterine horn was flushed with Ham's FlO medium 7 and the flushings from animals at the same stage of pregnancy were pooled, dialyzed against distilled water (at 4 o C) for 24 hr., and then lyophilized. The protein components were separated by gel filtration through Sephadex G-200 and determined quantitatively according to Lowry's procedure. 8 Six different protein fractions are identifiable by this procedure, although the relative amount of each fraction differs between 3- and 5-day pregnant animals (Fig. 1). The fractions were numbered I-VI, I being the highest molecular weight. By this system blastokinin is Fraction V. Each of the six fractions, as collected from 3-day and 5-day uteri, was tested alone and in combination against 3-day morulae and 5-day blastocysts in vitro. Total uterine fluid macromolecular compoReceived August 5, 1971; revised October 29, 1971. nents (from the unfractionated powder *Supported by NIH Grant R01 HD 04165 and AEC Contract AT(ll-1)-1597. obtained after lyophilization) from both t Present address: Max-Planck Institut Fur ages and serum macromolecular compoTierzucht- und Tiererniihrung, 3051 Mariensee tiber nents (also prepared by dialysis and lyoWunstorf, West Germany. philization of rabbit serum) were also :j: Present address: Department of Zoology and tested. Entomology, University of Tennessee, Knoxville, Tennessee. The basic culture technic has been de105
EL-BANNA AND DANIEL
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FIG. 1. Gel filtration through Sephadex G-200 of 3- and 5-day rabbit uterine fluid proteins. Each fraction used in these studies is designated by a roman numeral. The patterns are normalized to 1 mg. of total protein in each case.
scribed earlier 9 but was essentially as follows. Embryos were obtained by flushing the genital tracts from does on Day 3 or 5 post coitum with FlO solution that had been equilibrated in the culture incubator for 1 hr. before use. The embryos were then transferred, via capillary pipettes, into watch glasses (10 mm. diameter) containing 0.5-1 ml. of FlO medium with or without supplementation of the various uterine fluid components as described above. Five to 10 morulae (3 day) were cultured in the same watch glass. Five-day blastocysts were cultured individually for the growth experiments and in groups of 5 for the uptake experiments. The watch
Vol. 23
glasses were supported inside Petri dishes and the embryos were incubated at 38° C in an atmosphere of 5% CO 2 in air at saturation humidity. Four kinds of tests were conducted, designed to compare the effects of these protein and total macromolecular components on growth of the embryo and on the synthesis of deoxyribonucleic acid (DNA), RNA, and protein (as measured by uptake of radiolabeled nucleic acid precursors and amino acids). At least 10 embryos (and in some cases as many as 20 embryos) were used for each determination of each concentration of each component tested. Collectively, about 3000 rabbit embryos were used in these studies, and the uterine fluids obtained from at least 300 animals. For the growth experiments 3-day morulae were observed after 24, 48, and 72 hr. of incubation and scored for the number of embryos that had cavitated and exhibited expansion. The diameter of each 5-day blastocyst was measured with an ocular micrometer at the beginning of the culture period and every 24 hr. thereafter until termination at 72 hr. For the uptake experiments, blastocysts were first precultured in FlO for 1 hr. to wash their surfaces free of any adhering protein and/or to dilute it. Blastocysts were divided into groups so that their sizes varied minimally in a given set of experiments, although sizes varied considerably from one experiment to the next. The blastocysts were incubated as before (five to one culture dish containing 0.6 ml. of the medium) and removed for uptake determinations after 4 or 20 hr. exposure to the radioisotope. Each of the test media contained, in addition to their protein supplement, either 20 J.tCi./ml. [H 3 ] thymidine-methyl (specific activity 18 Ci./mM), 20 J.tCi./ml. [5-H 3 ] uridine (specific activity 11 Ci./ mM), or 10 J.tCi./ml. of [C 14 ] L-amino acid mixture (mixture of 15 amino acids, uniformly labeled).
February 1972
EFFECT OF PROTEIN FRACTIONS
Preparation of Embryos for Scintillation Counting
107
period, blastocysts were washed and transferred into individual test tubes containing 0.25 ml. of 0.25% a-amylase solution in 0.2 M sodium phosphate buffer pH 6.9 containing 0.006 M sodium chloride to remove the mucin coat. After 1 hr. at room temperature, the blastocysts were solubilized by the addition of 0.25 ml. of 2% SDS and 10 M urea solution and left for 30 min. at room temperature. The samples were then precipitated with 0.5 ml. of cold 0.05 M sodium pyrophosphate in 10% TCA and were placed in the cold overnight. The content of each tube was filtered on Millipore filter discs. The tubes were rinsed three times with a total volume of 15 ml. of cold 0.05 M pyrophosphate in 5% TCA, and the filter discs were washed with the resulting rinses. The filter discs were then washed with 10 ml. of hot (80-90° C) 5% TCA and then with 5 ml. of cold (0-4° C) 5% TCA and finally with 10 ml. of cold 70% ethanol. The filters were dried and prepared for counting as described above. A 0.1 ml. aliquot of the hot medium was processed as described to determine background. Statistical Treatment. The data were analyzed by Duncan's New Multiple Range Test with Kramer's extension to unequal samples. 11 This test was used because of the differences in sample size and the increase in embryonic deaths with prolonged culture. Significance at the 95% level is shown by a plus ( +) mark in appropriate columns of Tables 1, 2, and 3 when growth or uptake of precursor was better than the FlO controls. Where the data were not testable (usually because the controls did not survive) an expression of relative growth (i.e., poor, moderate, or good) was substituted.
Thymidine and Uridine. At the end of the incubation period the blastocysts were transferred through three washes of unlabeled FlO medium to dilute the unincorporated label, and then transferred into test tubes containing 0.05 ml. of a 1% pronase solution to remove the zona pellucidae. 10 The enzymatic reaction was terminated, after 1-2 min., by the addition of 0.5 ml. of freshly mixed sodium dodecyl sulfate (SDS) and urea at a final concentration of 1% SDS and 5 M urea. The blastocysts were left in the SDS-urea solution at room temperature for 30 min. The samples were precipitated with 1 ml. cold (4° C) 0.1 M sodium pyrophosphate (Matheson, Coleman, and Bell, Norwood, Ohio) in 10% trichloroacetic acid (TCA) and were left overnight in the cold. The precipitate was filtered on Millipore discs 2.4 em. in diameter supported on a Millipore filtering apparatus. The test tubes were rinsed three times with a total volume of 15 ml. cold 0.1 M sodium pyrophosphate in 10% TCA, and the filter discs washed, under gentle suction with the resulting rinses. The filter and funnel were washed with 10 ml. cold 70% ethanol, after which the filters were dried under an infrared lamp for 15 min. and then placed, precipitate up, in glass scintillation vials, and covered with 10 ml. scintillation fluid. The scintillation fluid was prepared by dissolving 8 gm. of Butyl-PBD (Beckman Instrument, Fullerton, Calif.) in 1 L. of toluene (J. T. Baker Chemical Co., Phillipsburg, N. J.). With every set of experiments, 0.1 ml. aliquot of the "hot" medium was transferred to a test tube and processed as described. The count of resulting filters was used to determine the background which RESULTS was then subtracted from the counts observed for the experimental groups. The results of these studies are shown Counting was performed in a Beckman in Tables 1, 2, and 3 and parts of the data Model LS-233 liquid scintillation counter. shown graphically in Figs. 2 and 3. AlAmino Acids. After the incubation though in many cases the data are highly
TABLE 1. Effect of Uterine Fluid Components on Growth of 3-Day Rabbit Morulae in Vitro Fractions from 3-day uteri
Concentration (mg. protein/ mi. FlO) Cavitation (%) Increase in diameter significantly better (at 95% level) than FlO controls after: 24 hr. 48 hr. 72 hr.
0.05 0.2 0.5 1 0.05 0.2 0.5 1
75 75 75 68
88 100 58 53
+ +
+
+ +
coc
VI
IV
0.05 0.2 0.5 1
0.05 0.2 0.5 1
0.05 0.2 0.5 1 0.05 0.2 0.5 1
100 92 100 100
87 94 93 93
93 100 85 89
+ + Not testable MG GG GG Not testable
v
III
II
I
Fractions
+
+ +
+
+ +
+ +
+
+ +
+
91 92 83 91
+
+
Not testable Not testable MG GG GG GG MG MG
+
+
UMC
SMC
0.5
0.5
90
100
100
+
+
+
+
+
+
+
+
FlO
80
trJ t"'
0, >
72 hr.
60 77 75 67
80 87 100 87
100 82 90 80
Not testable PG MG MG
Not GG Not GG
77 86 82 92
95 100 89 79
50 80 80 80
+ + Not testable GG MG
testable GG GG GG + + testable Not testable GG GG GG MG PG
~
t:l t:l
1Z
Fractions from 5-day uteri
Cavitation (%) Increase in diameter significantly better (at 95% level) than FlO controls after: 24 hr. 48 hr.
z z >
+ +
+ +
+ + +
+ +
100 78
100
100
+
+
+
+
+
+
+
+
*Key: COC, combination of all six fractions at their optimal concentrations; UMC, total uterine macromolecular components; SMC, total serum macromolecular components; GG, good growth; MG, moderate growth; PG, poor growth.
tri t"'
~ t;
TABLE 2. Effect of 5-Day Uterine Fluid Components on Growth of 5-Day Rabbit Blastocysts in Vitro* Fractions
I
Concentration (mg. protein/ 0.05 0.2 0.5 1 mi. FlO) Increase in diameter significantly better (at 95% level) than FlO controls after: 24 hr. 48 hr. 72 hr. Not testable PG MG MG PG *Key, same as in Table 1.
II
III
v
IV
VI
0.05 0.2 0.5 1 0.05 0.2 0.5 1 0.05 0.2 0.5 1 0.05 0.2 0.5 1 0.05 0.2 0.5 1
+ +
+
+ +
+ +
+
+ + + + + + +
coc
UMC
0.5
+ +
SMC
0.5
110
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EL-BANNA AND DANIEL
TABLE 3. Effect of 5-Day Uterine Fluid Components on Uptake of Nucleic Acid and Protein Precursors by 5-Day Rabbit Blastocysts in Vitro* II
lTI
IV
v
VI
coc
UMC
SMC
4 20
4 20
4 20
4 20
4 20
4 20
4 20
4 20
4 20
+ + +
+
+
+
Fractions
Exposure time in vitro (hr.)
Compared to FlO controls, significantly better uptake of: (at 95% level) H' Thymidine H' Uridine C 14 Amino acids
+
+ + +
*Key, same as in Table 1.
variable, we feel that they support the following generalizations: 1. FlO supplemented with any one of the fractions in right concentration or in combination supports better growth and blastulation of the morulae than FlO alone. Best results were obtained with Fractions II and III from 3-day fluids and Fractions III and V from 5-day fluids. 2. Over-all growth of 5-day blastocysts is improved in media supplemented with Fractions II, IV, V, or VI. 3. Thymidine uptake by blastocysts does not differ significantly from the controls in any of the media, but best average results were seen in embryos exposed to Fraction III for 4 hr. 4. For the short exposure time (4 hr.), best uridine uptake in 5-day embryos was promoted in Fractions I, II, and V; for the longer exposure period (20 hr.) Fractions I, IV, V, and VI were best. 5. Amino acid uptake by blastocyst stage embryos was dramatically accelerated after 4 hr. in Fraction V medium and to a lesser extent in Fraction I. Uptake was also significantly better in the total uterine macromolecular components and in the combined fractions for the 4-hr. period. By 20 hr., little difference is noted in the effect of any of the fractions on amino acid uptake, except that all of them show slight improvement over the controls. 6. Medium supplemented with all of
the fractions, each at its optimal concentration, never supported better growth or precursor uptake than the best individual fraction in optimal concentration. In fact, in regard to the uridine uptake studies, the results were poor, actually worse than those in the unsupplemented FlO controls. DISCUSSION
The type of experiments reported here are plagued with complications and certainly the variability of the results are ample evidence. This problem was discussed in another recent publication 3 and we feel compelled to quote part of that discussion as highly relevant to the present work. "Any attempt to analyze the relative effects of different substances at different concentrations on growth of cultured embryos is at best a frustrating experience. The size and growth in vivo of preimplantation stage embryos, even of sibling genotypes in the same uterus at the same time, varies considerably. With explantation to an artificial environment the variability is compounded. In spite of rigid efforts to keep the conditions of the culture techniques constant, individual embryos still respond differently to highly local variations in media, temperature, light, pH, oxygen concentration, condition of the
February 1972
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111
EFFECT OF PROTEIN FRACTIONS
at 0 hr
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Fraction ll
Fraction ill
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Fraction .ID
after 24 hr incubation after 48 hr after 72 hr
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Protein Concentration mg/ ml F10
FIG. 2. Growth of rabbit blastocysts in vitro in media supplemented with individual uterine protein fractions collected from rabbits on Day 5 post coitum.
glassware, etc. The best one can hope to do is to demonstrate general tendencies which may not survive strict statistical scrutiny. This is certainly the case when studying the effects of uterine proteins on rabbit blastocysts in vitro. Many of these proteins are already present in the blastocoelic fluid and presumably can act as a store to supply the cells' growth needs
for some time. And of course, the supply for larger blastocysts is greater than for smaller ones; not only in an absolute sense, but also in proportion to the cells available because of changes in the surface-volume ratio with increasing size of a sphere. Thus, the observations reported here cannot be considered to reflect absolute values, but at best only trends."
112
Vol. 23
EL-BANNA AND DANIEL
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Concentration of 5 days p c. Rabbit Uterine Protein Fract1ons ( mg/ml F10 ) FIG. 3. Incorporation of H'-thymidine, H'-uridine and C ''-amino acids by 5-day rabbit blastocysts cultured for 20 hr. in individual uterine protein fractions.
Since this work was initiated we have become more cognizant of the problem of maintaining the uterine proteins in an unaltered form during the preparative and experimental procedures and of the possibility that other bound molecules (e.g., see Arthur, Cowan, and Daniel 12) may be important to their biologic activity. These conditions further compound the problems of variability. Three-day morulae are stimulated by different fractions depending upon whether they come from fluids collected on Day 3 or Day 5. We feel that this must reflect the changes occuring in these fluids during this time; for example, Fraction V (blastokinin) is scarcely present in the secretions of a 3-day uterus but is the predominant component in the 5-day uterus. (See Fig. 1). The reader is also reminded that the fractions used merely represent
convenient separations according to molecular weight and that each fraction may be composed of one or many individual proteins. Changes, even of a major nature, in the proteins composing a particular fraction, would not necessarily be detectable by the molecular seiving technic. Furthermore, it must be realized that some contamination frequently occurs from one fraction to another during Sephadex gel filtration and that the activity of one fraction might therefore be reflected to some extent in an adjoining fraction. (It was not economically practical for us to employ extensive procedures for highly efficient purification of each fraction from the limited amount of material available to us.) It is interesting to note, that in the growth studies the optimal concentration of a given fraction was almost always in
February 1972
EFFECT OF PROTEIN FRACTIONS
the concentration of 0.2 or 0.5 mg./ml. This agrees precisely with the earlier reports 1• 3 and is reflective of the results reported from studies of nutritional components for cell culture media. It may also help explain the difficulty in culturing blastocyst stage embryos; the concentration of one such component in unreplenished medium would change continuously by virtue of its being metabolized and/or denatured and the specific requirements for that component by an embryo that is growing and differentiating must also be changing. If one tabulates the effects of each fraction on all of the parameters studied it becomes apparent that different fractions predominate in different ways. Fraction I stimulates uridine uptake and, to a lesser degree, amino acid uptake. Fraction II also stimulates uridine uptake and some embryonic growth. Fraction III stimulates growth of morulae and the best average thymidine uptake. Fraction IV stimulates morulae growth and uridine uptake. Fraction V is effective in all categories except that it does not differ significantly from the other fractions in respect to its influence on thymidine uptake. Fraction VI stimulates blastocyst growth. The results reported here, considered in general, agree fairly well with the earlier reported studies on cavitation of morulae, and growth and precursor uptake promoting effects of blastokinin on blastocysts, and they further establish that other of the uterine protein fractions also influence the preimplantation rabbit embryo. DanieP reported that Fractions III, IV, and V promoted best consistent growth of 5-day blastocysts. The studies reported here seem to implicate Fractions II, IV, V, and VI; Fraction III shows little effect in promoting over-all growth (however, it was not studied at the high concentration at which Daniel found best results). Fraction III was most effective in promoting cavita-
113
tion and growth from the morula stage. Thymidine uptake does not seem to be improved significantly over the controls by any fraction (with the possible exception of Fraction III). This is not surprising when one recalls that the mitotic index for the cells of early rabbit embryos is about 4.42%, 13 and that even after 18 hr. culture in serum-supplemented medium the embryo still has a mitotic index that averages 4.21 %. 4 Mitotic index ranges between 4 and 5% in a number of mammal embryos during their most active phases (e.g., 14 • 15). Thus, this level would seem to be close to the limit of the replicative capacity of these cells and probably cannot be readily exceeded. This limit would be reflected in DNA synthesis and therefore one would expect to see a fairly constant level, regardless of the kinds of stimuli used. Uridine uptake, presumably as a measure of RNA synthesis, is stimulated by four of the six fractions after 20 hr. exposure. Amino acid uptake, as a measure of protein synthesis, is markedly stimulated by two fractions (I and V) after 4 hr. exposure, and is better than the controls after both 4 and 20 hr. exposure in media containing any one of the fractions. Thus, it appears that all of the uterine proteins may be desirable (possibly essential) for development of rabbit blastocysts. To determine whether their actions are highly specific will require extensive experiments beyond those reported here. In all of these studies, the action of Fraction V from the 5-day uterus predominates. It stimulates growth of morulae and blastocysts, uridine uptake and best amino acid uptake by blastocysts after short exposure. These latter observations agree with those reported by Gulyas, Daniel, and Krishnan 6 and Manes. 5 Also, this fraction is separable by the Sephadex procedure in relatively pure form 16 so that one need not be bothered by concern for contamination. Therefore, we continue to
114
EL-BANNA AND DANIEL
feel convinced that whatever the function of other uterine proteins, blastokinin has a special importance for embryogenesis of the rabbit blastocyst. SUMMARY
Each of the six different molecular weight fractions of the proteinaceous components composing rabbit uterine fluids was isolated by Sephadex gel filtration. Culture media were supplemented with these fractions, individually and in combinations, in an attempt to appraise their effects on growth of rabbit morulae and blastocysts, and on the uptake of radiolabeled precursors for nucleic acids and proteins. Comparisons were made with embryos incubated in defined medium and media supplemented with the macromolecular components of serum. In general, media supplemented with any of the fractions or serum components support better growth and amino acid or uridine uptake for one stage embryo than unsupplemented medium. A combination of all fractions at their optimal concentrations supports no better growth than the best individual fraction. Thymidine uptake stays relatively constant in all media, presumably reflecting a constant, unexceedable level of DNA synthesis and mitotis. Fraction V, which is blastokinin, was the most effective promoter of blastocyst growth and incorporation of uridine and amino acids. REFERENCES 1. KRISHNAN, R. S., AND DANIEL, J. C. "Blastokinin": inducer and regulator of blastocyst development in the rabbit uterus. Science 158:490, 1967. 2. BEIER, H. M. Uteroglobin: A hormone sensitive
Vol. 23
endometrial protein involved in blastocyst de· velopment. Biochim Biophys Acta 160:289, 1968. 3. DANIEL, J. C. Uterine proteins and embryonic development. Advances Biosci 6:191, 1971. 4. DANIEL, J. C., AND KRISHNAN, R. S. Studies on the relationship between uterine fluid compo· nents and the diapausing state of blastocysts from mammals having delayed implantation. J Exp Zool 172:267, 1969. 5. MANES, C. A study of protein synthesis during the preimplantation development of the rabbit embryo. Ph.D. Thesis-University of Colorado. Dissertation Abstracts 29:3546b, 1968. 6. GULYAS, B. J., DANIEL, J. C., AND KRISHNAN, R. S. Incorporation of labelled nucleosides in vitro by rabbit and mink blastocysts in the presence of blastokinin or serum. J Reprod Fertil 20:255, 1969. 7. HAM, R. G. An improved nutrient solution for diploid Chinese hamster and human cell lines. Exp Cell Res 29:515, 1963. 8. LowRY, 0. H., RosEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. Protein measurement with the folin phenol reagent. J Biol Chem 193:265, 1951. 9. DANIEL, J. C. Studies on the growth of 5-day-old rabbit blastocysts in vitro. J Embryo/ Exp Morph 13:83, 1965. 10. MINTZ, B. Experimental study of the developing mammalian egg: removal of the zona pellucida. Science 138:594, 1962. 11. KRAMER, C. Y. Extension of multiple range tests to group means with unequal numbers of replications. Biometrics 12:307, 1956. 12. ARTHUR, A. T., COWAN, B. D., AND DANIEL, J. C. Steroid binding to blastokinin. Fertil Steril 23:85, 1972 13. DANIEL, J. C. Early growth of rabbit trophoblast. A mer Natur 98:85, 1964. 14. DANIEL, J. C. Coincidence of embryonic growth and uterine protein in the ferret. J Embryo/ Exp Morph 24:305, 1970. 15. DANIEL, J. C. Growth of the preimplantation embryo of the northern fur seal and its correlation with changes in uterine protein. Develop Bio/26:316, 1971. 16. KRISHNAN, R. S., AND DANIEL, J. C. Composition of blastokinin from the rabbit uterus. Biochim Biophys Acta 168:579, 1968.
FERTILITY AND STERILITY
Copyright © 1972 by The Williams & Wilkins Co.
THE EFFECT OF PROTEIN FRACTIONS FROM RABBIT UTERINE FLUIDS ON EMBRYO GROWTH AND UPTAKE OF NUCLEIC ACID AND PROTEIN PRECURSORS* AMIN A. EL-BANNA, PH.D., t
AND
JOSEPH C. DANIEL, JR., PH.D.:j:
Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, Colorado
Specific proteins appear in the uterine secretions of mammals during pregnancy and at times that coincide with specific developmental stages of their embryos. Such coincidence has prompted investigators to assume that dependent interrelationships exist: primarily that one or more of the proteins are essential for growth and/or differentiation of the corresponding stage embryo. 1 - 3 One especially prominent protein is secreted by the uterus of the rabbit during the period when the embryos are free-living, unattached blastocysts. When tested in vitro, this protein, "blastokinin," stimulated blastulation of morulae, 1 growth of blastocysts, 3 new mitotic activity in "dormant" blastocysts, • and synthesis of protein 5 and ribonucleic acid (RNA) 6 as measured by uptake of radiolabeled precursors. To the best of our knowledge this is the only uterine protein that has been tested for its effects on embryos even though as many as 18 separate proteins (some of serum origin) have been reported to exist in the rabbit uterus. By G-200 Sephadex gel filtration of rabbit uterine proteins collected in early pregnancy, prior to implantation, six different molecular weight
fractions can be identified and isolated: The present report concerns the results of studies designed to test the comparative effects of each of these six fractions on rabbit morulae and blastocysts in vitro. METHODS AND MATERIALS
Uterine fluids were collected from rabbits on the 3rd and 5th day postcoitum. Each isolated uterine horn was flushed with Ham's FlO medium 7 and the flushings from animals at the same stage of pregnancy were pooled, dialyzed against distilled water (at 4 o C) for 24 hr., and then lyophilized. The protein components were separated by gel filtration through Sephadex G-200 and determined quantitatively according to Lowry's procedure. 8 Six different protein fractions are identifiable by this procedure, although the relative amount of each fraction differs between 3- and 5-day pregnant animals (Fig. 1). The fractions were numbered I-VI, I being the highest molecular weight. By this system blastokinin is Fraction V. Each of the six fractions, as collected from 3-day and 5-day uteri, was tested alone and in combination against 3-day morulae and 5-day blastocysts in vitro. Total uterine fluid macromolecular compoReceived August 5, 1971; revised October 29, 1971. nents (from the unfractionated powder *Supported by NIH Grant R01 HD 04165 and AEC Contract AT(ll-1)-1597. obtained after lyophilization) from both t Present address: Max-Planck Institut Fur ages and serum macromolecular compoTierzucht- und Tiererniihrung, 3051 Mariensee tiber nents (also prepared by dialysis and lyoWunstorf, West Germany. philization of rabbit serum) were also :j: Present address: Department of Zoology and tested. Entomology, University of Tennessee, Knoxville, Tennessee. The basic culture technic has been de105
EL-BANNA AND DANIEL
106 035
ill 0 30
3 DAY 0 25
IV
010
"
E 0
"',_
0 05
0 ~
~
0
0
u
0 25
~
0
0 20
015
010
005
0
20
40
60
80
Fract1on
Number
FIG. 1. Gel filtration through Sephadex G-200 of 3- and 5-day rabbit uterine fluid proteins. Each fraction used in these studies is designated by a roman numeral. The patterns are normalized to 1 mg. of total protein in each case.
scribed earlier 9 but was essentially as follows. Embryos were obtained by flushing the genital tracts from does on Day 3 or 5 post coitum with FlO solution that had been equilibrated in the culture incubator for 1 hr. before use. The embryos were then transferred, via capillary pipettes, into watch glasses (10 mm. diameter) containing 0.5-1 ml. of FlO medium with or without supplementation of the various uterine fluid components as described above. Five to 10 morulae (3 day) were cultured in the same watch glass. Five-day blastocysts were cultured individually for the growth experiments and in groups of 5 for the uptake experiments. The watch
Vol. 23
glasses were supported inside Petri dishes and the embryos were incubated at 38° C in an atmosphere of 5% CO 2 in air at saturation humidity. Four kinds of tests were conducted, designed to compare the effects of these protein and total macromolecular components on growth of the embryo and on the synthesis of deoxyribonucleic acid (DNA), RNA, and protein (as measured by uptake of radiolabeled nucleic acid precursors and amino acids). At least 10 embryos (and in some cases as many as 20 embryos) were used for each determination of each concentration of each component tested. Collectively, about 3000 rabbit embryos were used in these studies, and the uterine fluids obtained from at least 300 animals. For the growth experiments 3-day morulae were observed after 24, 48, and 72 hr. of incubation and scored for the number of embryos that had cavitated and exhibited expansion. The diameter of each 5-day blastocyst was measured with an ocular micrometer at the beginning of the culture period and every 24 hr. thereafter until termination at 72 hr. For the uptake experiments, blastocysts were first precultured in FlO for 1 hr. to wash their surfaces free of any adhering protein and/or to dilute it. Blastocysts were divided into groups so that their sizes varied minimally in a given set of experiments, although sizes varied considerably from one experiment to the next. The blastocysts were incubated as before (five to one culture dish containing 0.6 ml. of the medium) and removed for uptake determinations after 4 or 20 hr. exposure to the radioisotope. Each of the test media contained, in addition to their protein supplement, either 20 J.tCi./ml. [H 3 ] thymidine-methyl (specific activity 18 Ci./mM), 20 J.tCi./ml. [5-H 3 ] uridine (specific activity 11 Ci./ mM), or 10 J.tCi./ml. of [C 14 ] L-amino acid mixture (mixture of 15 amino acids, uniformly labeled).
February 1972
EFFECT OF PROTEIN FRACTIONS
Preparation of Embryos for Scintillation Counting
107
period, blastocysts were washed and transferred into individual test tubes containing 0.25 ml. of 0.25% a-amylase solution in 0.2 M sodium phosphate buffer pH 6.9 containing 0.006 M sodium chloride to remove the mucin coat. After 1 hr. at room temperature, the blastocysts were solubilized by the addition of 0.25 ml. of 2% SDS and 10 M urea solution and left for 30 min. at room temperature. The samples were then precipitated with 0.5 ml. of cold 0.05 M sodium pyrophosphate in 10% TCA and were placed in the cold overnight. The content of each tube was filtered on Millipore filter discs. The tubes were rinsed three times with a total volume of 15 ml. of cold 0.05 M pyrophosphate in 5% TCA, and the filter discs were washed with the resulting rinses. The filter discs were then washed with 10 ml. of hot (80-90° C) 5% TCA and then with 5 ml. of cold (0-4° C) 5% TCA and finally with 10 ml. of cold 70% ethanol. The filters were dried and prepared for counting as described above. A 0.1 ml. aliquot of the hot medium was processed as described to determine background. Statistical Treatment. The data were analyzed by Duncan's New Multiple Range Test with Kramer's extension to unequal samples. 11 This test was used because of the differences in sample size and the increase in embryonic deaths with prolonged culture. Significance at the 95% level is shown by a plus ( +) mark in appropriate columns of Tables 1, 2, and 3 when growth or uptake of precursor was better than the FlO controls. Where the data were not testable (usually because the controls did not survive) an expression of relative growth (i.e., poor, moderate, or good) was substituted.
Thymidine and Uridine. At the end of the incubation period the blastocysts were transferred through three washes of unlabeled FlO medium to dilute the unincorporated label, and then transferred into test tubes containing 0.05 ml. of a 1% pronase solution to remove the zona pellucidae. 10 The enzymatic reaction was terminated, after 1-2 min., by the addition of 0.5 ml. of freshly mixed sodium dodecyl sulfate (SDS) and urea at a final concentration of 1% SDS and 5 M urea. The blastocysts were left in the SDS-urea solution at room temperature for 30 min. The samples were precipitated with 1 ml. cold (4° C) 0.1 M sodium pyrophosphate (Matheson, Coleman, and Bell, Norwood, Ohio) in 10% trichloroacetic acid (TCA) and were left overnight in the cold. The precipitate was filtered on Millipore discs 2.4 em. in diameter supported on a Millipore filtering apparatus. The test tubes were rinsed three times with a total volume of 15 ml. cold 0.1 M sodium pyrophosphate in 10% TCA, and the filter discs washed, under gentle suction with the resulting rinses. The filter and funnel were washed with 10 ml. cold 70% ethanol, after which the filters were dried under an infrared lamp for 15 min. and then placed, precipitate up, in glass scintillation vials, and covered with 10 ml. scintillation fluid. The scintillation fluid was prepared by dissolving 8 gm. of Butyl-PBD (Beckman Instrument, Fullerton, Calif.) in 1 L. of toluene (J. T. Baker Chemical Co., Phillipsburg, N. J.). With every set of experiments, 0.1 ml. aliquot of the "hot" medium was transferred to a test tube and processed as described. The count of resulting filters was used to determine the background which RESULTS was then subtracted from the counts observed for the experimental groups. The results of these studies are shown Counting was performed in a Beckman in Tables 1, 2, and 3 and parts of the data Model LS-233 liquid scintillation counter. shown graphically in Figs. 2 and 3. AlAmino Acids. After the incubation though in many cases the data are highly
TABLE 1. Effect of Uterine Fluid Components on Growth of 3-Day Rabbit Morulae in Vitro Fractions from 3-day uteri
Concentration (mg. protein/ mi. FlO) Cavitation (%) Increase in diameter significantly better (at 95% level) than FlO controls after: 24 hr. 48 hr. 72 hr.
0.05 0.2 0.5 1 0.05 0.2 0.5 1
75 75 75 68
88 100 58 53
+ +
+
+ +
coc
VI
IV
0.05 0.2 0.5 1
0.05 0.2 0.5 1
0.05 0.2 0.5 1 0.05 0.2 0.5 1
100 92 100 100
87 94 93 93
93 100 85 89
+ + Not testable MG GG GG Not testable
v
III
II
I
Fractions
+
+ +
+
+ +
+ +
+
+ +
+
91 92 83 91
+
+
Not testable Not testable MG GG GG GG MG MG
+
+
UMC
SMC
0.5
0.5
90
100
100
+
+
+
+
+
+
+
+
FlO
80
trJ t"'
0, >
72 hr.
60 77 75 67
80 87 100 87
100 82 90 80
Not testable PG MG MG
Not GG Not GG
77 86 82 92
95 100 89 79
50 80 80 80
+ + Not testable GG MG
testable GG GG GG + + testable Not testable GG GG GG MG PG
~
t:l t:l
1Z
Fractions from 5-day uteri
Cavitation (%) Increase in diameter significantly better (at 95% level) than FlO controls after: 24 hr. 48 hr.
z z >
+ +
+ +
+ + +
+ +
100 78
100
100
+
+
+
+
+
+
+
+
*Key: COC, combination of all six fractions at their optimal concentrations; UMC, total uterine macromolecular components; SMC, total serum macromolecular components; GG, good growth; MG, moderate growth; PG, poor growth.
tri t"'
~ t;
TABLE 2. Effect of 5-Day Uterine Fluid Components on Growth of 5-Day Rabbit Blastocysts in Vitro* Fractions
I
Concentration (mg. protein/ 0.05 0.2 0.5 1 mi. FlO) Increase in diameter significantly better (at 95% level) than FlO controls after: 24 hr. 48 hr. 72 hr. Not testable PG MG MG PG *Key, same as in Table 1.
II
III
v
IV
VI
0.05 0.2 0.5 1 0.05 0.2 0.5 1 0.05 0.2 0.5 1 0.05 0.2 0.5 1 0.05 0.2 0.5 1
+ +
+
+ +
+ +
+
+ + + + + + +
coc
UMC
0.5
+ +
SMC
0.5
110
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EL-BANNA AND DANIEL
TABLE 3. Effect of 5-Day Uterine Fluid Components on Uptake of Nucleic Acid and Protein Precursors by 5-Day Rabbit Blastocysts in Vitro* II
lTI
IV
v
VI
coc
UMC
SMC
4 20
4 20
4 20
4 20
4 20
4 20
4 20
4 20
4 20
+ + +
+
+
+
Fractions
Exposure time in vitro (hr.)
Compared to FlO controls, significantly better uptake of: (at 95% level) H' Thymidine H' Uridine C 14 Amino acids
+
+ + +
*Key, same as in Table 1.
variable, we feel that they support the following generalizations: 1. FlO supplemented with any one of the fractions in right concentration or in combination supports better growth and blastulation of the morulae than FlO alone. Best results were obtained with Fractions II and III from 3-day fluids and Fractions III and V from 5-day fluids. 2. Over-all growth of 5-day blastocysts is improved in media supplemented with Fractions II, IV, V, or VI. 3. Thymidine uptake by blastocysts does not differ significantly from the controls in any of the media, but best average results were seen in embryos exposed to Fraction III for 4 hr. 4. For the short exposure time (4 hr.), best uridine uptake in 5-day embryos was promoted in Fractions I, II, and V; for the longer exposure period (20 hr.) Fractions I, IV, V, and VI were best. 5. Amino acid uptake by blastocyst stage embryos was dramatically accelerated after 4 hr. in Fraction V medium and to a lesser extent in Fraction I. Uptake was also significantly better in the total uterine macromolecular components and in the combined fractions for the 4-hr. period. By 20 hr., little difference is noted in the effect of any of the fractions on amino acid uptake, except that all of them show slight improvement over the controls. 6. Medium supplemented with all of
the fractions, each at its optimal concentration, never supported better growth or precursor uptake than the best individual fraction in optimal concentration. In fact, in regard to the uridine uptake studies, the results were poor, actually worse than those in the unsupplemented FlO controls. DISCUSSION
The type of experiments reported here are plagued with complications and certainly the variability of the results are ample evidence. This problem was discussed in another recent publication 3 and we feel compelled to quote part of that discussion as highly relevant to the present work. "Any attempt to analyze the relative effects of different substances at different concentrations on growth of cultured embryos is at best a frustrating experience. The size and growth in vivo of preimplantation stage embryos, even of sibling genotypes in the same uterus at the same time, varies considerably. With explantation to an artificial environment the variability is compounded. In spite of rigid efforts to keep the conditions of the culture techniques constant, individual embryos still respond differently to highly local variations in media, temperature, light, pH, oxygen concentration, condition of the
February 1972
3.4
2.6
0 f2l 0 f3
111
EFFECT OF PROTEIN FRACTIONS
at 0 hr
Fraction I
Fraction ll
Fraction ill
Fraction N
Fraction Y.
Fraction .ID
after 24 hr incubation after 48 hr after 72 hr
1.8
1.0 w
en -ti
3.4
E E c
2.6
0
~
.0
E
w
I .8
Q)
.::;
....0 ...
~
1.0
Q)
E 0
0
3.4
2.6
I .8
1.0
0.2
0.05
0
1.0
05
02
0.05
0
Protein Concentration mg/ ml F10
FIG. 2. Growth of rabbit blastocysts in vitro in media supplemented with individual uterine protein fractions collected from rabbits on Day 5 post coitum.
glassware, etc. The best one can hope to do is to demonstrate general tendencies which may not survive strict statistical scrutiny. This is certainly the case when studying the effects of uterine proteins on rabbit blastocysts in vitro. Many of these proteins are already present in the blastocoelic fluid and presumably can act as a store to supply the cells' growth needs
for some time. And of course, the supply for larger blastocysts is greater than for smaller ones; not only in an absolute sense, but also in proportion to the cells available because of changes in the surface-volume ratio with increasing size of a sphere. Thus, the observations reported here cannot be considered to reflect absolute values, but at best only trends."
112
Vol. 23
EL-BANNA AND DANIEL
V>
"0
Q)
lilllot~; I -
I
'
c I
~
I(')
I(')
200
"'c:
c
180
0:: Q)
~
£
-!1
160
,-
IT
'o
" ~ .0
80
'
60
120
60
40
100
40
20
80
20
140
80
I
E w
::;:
m
Q_
0
~
h I 0.3
rt-r~h
~
}\:I
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n 0.3
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w
1ll 0.5
~I
TI. ::~
!~::
I
IlL
Y.
:srr
FlO
0.3
0.3
0.3
(control)
Concentration of 5 days p c. Rabbit Uterine Protein Fract1ons ( mg/ml F10 ) FIG. 3. Incorporation of H'-thymidine, H'-uridine and C ''-amino acids by 5-day rabbit blastocysts cultured for 20 hr. in individual uterine protein fractions.
Since this work was initiated we have become more cognizant of the problem of maintaining the uterine proteins in an unaltered form during the preparative and experimental procedures and of the possibility that other bound molecules (e.g., see Arthur, Cowan, and Daniel 12) may be important to their biologic activity. These conditions further compound the problems of variability. Three-day morulae are stimulated by different fractions depending upon whether they come from fluids collected on Day 3 or Day 5. We feel that this must reflect the changes occuring in these fluids during this time; for example, Fraction V (blastokinin) is scarcely present in the secretions of a 3-day uterus but is the predominant component in the 5-day uterus. (See Fig. 1). The reader is also reminded that the fractions used merely represent
convenient separations according to molecular weight and that each fraction may be composed of one or many individual proteins. Changes, even of a major nature, in the proteins composing a particular fraction, would not necessarily be detectable by the molecular seiving technic. Furthermore, it must be realized that some contamination frequently occurs from one fraction to another during Sephadex gel filtration and that the activity of one fraction might therefore be reflected to some extent in an adjoining fraction. (It was not economically practical for us to employ extensive procedures for highly efficient purification of each fraction from the limited amount of material available to us.) It is interesting to note, that in the growth studies the optimal concentration of a given fraction was almost always in
February 1972
EFFECT OF PROTEIN FRACTIONS
the concentration of 0.2 or 0.5 mg./ml. This agrees precisely with the earlier reports 1• 3 and is reflective of the results reported from studies of nutritional components for cell culture media. It may also help explain the difficulty in culturing blastocyst stage embryos; the concentration of one such component in unreplenished medium would change continuously by virtue of its being metabolized and/or denatured and the specific requirements for that component by an embryo that is growing and differentiating must also be changing. If one tabulates the effects of each fraction on all of the parameters studied it becomes apparent that different fractions predominate in different ways. Fraction I stimulates uridine uptake and, to a lesser degree, amino acid uptake. Fraction II also stimulates uridine uptake and some embryonic growth. Fraction III stimulates growth of morulae and the best average thymidine uptake. Fraction IV stimulates morulae growth and uridine uptake. Fraction V is effective in all categories except that it does not differ significantly from the other fractions in respect to its influence on thymidine uptake. Fraction VI stimulates blastocyst growth. The results reported here, considered in general, agree fairly well with the earlier reported studies on cavitation of morulae, and growth and precursor uptake promoting effects of blastokinin on blastocysts, and they further establish that other of the uterine protein fractions also influence the preimplantation rabbit embryo. DanieP reported that Fractions III, IV, and V promoted best consistent growth of 5-day blastocysts. The studies reported here seem to implicate Fractions II, IV, V, and VI; Fraction III shows little effect in promoting over-all growth (however, it was not studied at the high concentration at which Daniel found best results). Fraction III was most effective in promoting cavita-
113
tion and growth from the morula stage. Thymidine uptake does not seem to be improved significantly over the controls by any fraction (with the possible exception of Fraction III). This is not surprising when one recalls that the mitotic index for the cells of early rabbit embryos is about 4.42%, 13 and that even after 18 hr. culture in serum-supplemented medium the embryo still has a mitotic index that averages 4.21 %. 4 Mitotic index ranges between 4 and 5% in a number of mammal embryos during their most active phases (e.g., 14 • 15). Thus, this level would seem to be close to the limit of the replicative capacity of these cells and probably cannot be readily exceeded. This limit would be reflected in DNA synthesis and therefore one would expect to see a fairly constant level, regardless of the kinds of stimuli used. Uridine uptake, presumably as a measure of RNA synthesis, is stimulated by four of the six fractions after 20 hr. exposure. Amino acid uptake, as a measure of protein synthesis, is markedly stimulated by two fractions (I and V) after 4 hr. exposure, and is better than the controls after both 4 and 20 hr. exposure in media containing any one of the fractions. Thus, it appears that all of the uterine proteins may be desirable (possibly essential) for development of rabbit blastocysts. To determine whether their actions are highly specific will require extensive experiments beyond those reported here. In all of these studies, the action of Fraction V from the 5-day uterus predominates. It stimulates growth of morulae and blastocysts, uridine uptake and best amino acid uptake by blastocysts after short exposure. These latter observations agree with those reported by Gulyas, Daniel, and Krishnan 6 and Manes. 5 Also, this fraction is separable by the Sephadex procedure in relatively pure form 16 so that one need not be bothered by concern for contamination. Therefore, we continue to
114
EL-BANNA AND DANIEL
feel convinced that whatever the function of other uterine proteins, blastokinin has a special importance for embryogenesis of the rabbit blastocyst. SUMMARY
Each of the six different molecular weight fractions of the proteinaceous components composing rabbit uterine fluids was isolated by Sephadex gel filtration. Culture media were supplemented with these fractions, individually and in combinations, in an attempt to appraise their effects on growth of rabbit morulae and blastocysts, and on the uptake of radiolabeled precursors for nucleic acids and proteins. Comparisons were made with embryos incubated in defined medium and media supplemented with the macromolecular components of serum. In general, media supplemented with any of the fractions or serum components support better growth and amino acid or uridine uptake for one stage embryo than unsupplemented medium. A combination of all fractions at their optimal concentrations supports no better growth than the best individual fraction. Thymidine uptake stays relatively constant in all media, presumably reflecting a constant, unexceedable level of DNA synthesis and mitotis. Fraction V, which is blastokinin, was the most effective promoter of blastocyst growth and incorporation of uridine and amino acids. REFERENCES 1. KRISHNAN, R. S., AND DANIEL, J. C. "Blastokinin": inducer and regulator of blastocyst development in the rabbit uterus. Science 158:490, 1967. 2. BEIER, H. M. Uteroglobin: A hormone sensitive
Vol. 23
endometrial protein involved in blastocyst de· velopment. Biochim Biophys Acta 160:289, 1968. 3. DANIEL, J. C. Uterine proteins and embryonic development. Advances Biosci 6:191, 1971. 4. DANIEL, J. C., AND KRISHNAN, R. S. Studies on the relationship between uterine fluid compo· nents and the diapausing state of blastocysts from mammals having delayed implantation. J Exp Zool 172:267, 1969. 5. MANES, C. A study of protein synthesis during the preimplantation development of the rabbit embryo. Ph.D. Thesis-University of Colorado. Dissertation Abstracts 29:3546b, 1968. 6. GULYAS, B. J., DANIEL, J. C., AND KRISHNAN, R. S. Incorporation of labelled nucleosides in vitro by rabbit and mink blastocysts in the presence of blastokinin or serum. J Reprod Fertil 20:255, 1969. 7. HAM, R. G. An improved nutrient solution for diploid Chinese hamster and human cell lines. Exp Cell Res 29:515, 1963. 8. LowRY, 0. H., RosEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. Protein measurement with the folin phenol reagent. J Biol Chem 193:265, 1951. 9. DANIEL, J. C. Studies on the growth of 5-day-old rabbit blastocysts in vitro. J Embryo/ Exp Morph 13:83, 1965. 10. MINTZ, B. Experimental study of the developing mammalian egg: removal of the zona pellucida. Science 138:594, 1962. 11. KRAMER, C. Y. Extension of multiple range tests to group means with unequal numbers of replications. Biometrics 12:307, 1956. 12. ARTHUR, A. T., COWAN, B. D., AND DANIEL, J. C. Steroid binding to blastokinin. Fertil Steril 23:85, 1972 13. DANIEL, J. C. Early growth of rabbit trophoblast. A mer Natur 98:85, 1964. 14. DANIEL, J. C. Coincidence of embryonic growth and uterine protein in the ferret. J Embryo/ Exp Morph 24:305, 1970. 15. DANIEL, J. C. Growth of the preimplantation embryo of the northern fur seal and its correlation with changes in uterine protein. Develop Bio/26:316, 1971. 16. KRISHNAN, R. S., AND DANIEL, J. C. Composition of blastokinin from the rabbit uterus. Biochim Biophys Acta 168:579, 1968.