Printed in Sweden Copyright 0 1975 by Academic Press, Inc. All rights of wproduction in any form wserwd
Experimental
EFFECTS
OF LOW
ACID
AND
Cell Research 90 (1975) 73-78
TEMPERATURE
PROTEIN
SYNTHESIS
G. B. ANDERSON1 Department
of Animal
Science,
UPON
SUBSEQUENT
OF RABBIT
NUCLEIC
EMBRYOS
and R. H. FOOTE
and Division of Biological Ithaca, N. Y. 14850, USA
Sciences,
Cornell
University,
SUMMARY Two-cell rabbit embryos, which were maintained without cleavage for 24 h at lO”C, and then allowed to develop into blastocysts in vivo or in a completely defined medium at 37”C, incorporated aH-uridine into RNA and WXeucine into protein at rates comparable to blastocysts not previously exposed to 10°C temperature. Thus, temporary suppression of development did not impair nucleic acid and protein synthesis by embryos developing into blastocysts. Blastocysts which developed in vivo incorporated more 3H-thymidine into DNA, 3H-uridine into RNA and 14C-leucine into protein than did blastocysts which developed in vitro. These differences probably reflect not only differences in blastocyst size and number of cells, but also differences in endogenous precursor pools.
Despite widespread manipulation of mammalian embryos in vitro little is known about effects of cooling embryos on subsequent metabolism. Recent studies showed that temporary inhibition of development of rabbit embryos by holding at 10°C for 24 h had no effect upon subsequent pyruvate or glucose utilization when the embryos were rewarmed to 37°C [l]. These findings suggested that embryo death following low temperature stress was not due to an induced inability of the embryo to obtain energy for development. The experiments reported in this paper were designed to determine the rates of incorporation of labeled precursors into DNA, RNA and protein by rabbit blastocysts which had been stored in the two-cell stage at 10°C for 24 h. These were compared with incorporation rates of blastocysts which were not stored at 10°C. 1 Present address: Department of Animal Science, University of California, Davis, Calif. 95616, USA.
MATERIALS
AND METHODS
DNA synthesis Two-cell embryos were collected from superovulated Dutch-Belted rabbits 26 h after LH administration, pooled and then randomly assigned to treatment groups to compare DNA synthesis in vitro and in ;ivo- following- inhibition at 10°C. Embryos were cultured into blastocysts in BSM II, a defined culture medium [4], for 94 h at 37°C (treatment I) or stored for 24 h at 10°C in BSM II plus 4 % Promine-D (w/v). a soybean protein, as previously described [l]. Foll&: ing storage at 10°C embryos were either cultured at 37°C for 94 h (II) or transferred to the oviducts of pseudopregnant females and recollected from the uteri 94 h after transfer (III). Treatment IV consisted of blastocysts collected from the uteri of superovulated females 120 h after LH administration. Since the embryos in treatments II and III did not divide while being held at 10°C in the two-cell stage, blastocysts from all treatments were assumed to represent embryos having undergone 120 h of development. Diameter measurements were determined on morphologically normal blastocysts from each of the treatment groups at the end of the development period. Blastocysts were incubated at 37°C in 0.2 ml BSM II with 3H-methylthymidine (6.7 Ci/mmole, New England Nuclear) added to achieve an activity of 10 ,&i/ml. After incubation in the labeled medium for 1 h, blastocysts were washed 4 times in BSM II to remove unincorporated 3H-thymidine. Following the fourth wash the embryos were moved to a watch Exptl
Cell Res 90 (1975)
74
Anderson and Foote
Fig. I. Autoradiograph of a 120-h rabbit blastocyst (430 x ). Most nuclei which incorporated 3H-thymidine during the incubation period are extensively covered with reduced silver grains. Non-labeled cells were stained bright blue with Harris hematoxylin, but appear gray in the photograph. The arrow indicates a cell in anaphase which was unlabeled, as expected.
glass containing 70% ethanol for 1 to 2 h. The alcohol treatment fixed the embryos, prevented them from synthesizing more DNA and also tended to loosen the intercell matrix so that individual cells saread to form a monolayer when the embryo was squashed on a glass slide. Embryos were then exnosed to a 0.5 % solution of pronase-for 10 to 30 set-followed quickly by a wash with nhvsioloaical saline. Blastocvsts were soread on clean; gelatin-coated glass slides and embryosquashcs ureoared as described bv Sanval & Meyer 16. 71. The slides were coated with Kodak NTB3 nuclear emulsion using a roller technique [S]. They were exposed for 5 days at 4°C followed by development and staining with Harris’ hematoxvlin. All nucl4 were stained blue, but nuclei which incorporated $H-thymidine were labeled with overlying reduced silver grains (fig. 1). The numbers of labeled and unlabeled cells were counted at 100 x magnification and the total number of cells per blastocyst and the percentage of total cells that were labeled were calculated. This latter parameter was used to compare the relative mitotic activity in embryos from the various treatment groups. An analysis of variance for unequal subclasses was used to test treatment differences and Tukey’s hsd test was used to test for differences among treatment means.
RNA synthesis Two-cell embryos were collected as for the DNA studies and assigned to 1 of 4 treatment groups. Treatments are indicated in table 2. Treatments I, II, Exptl
Cell Res 90 (1975)
1V and V correspond to treatments I-IV in the previous experiment (table 1). In addition, collected embryos which were not held at 10°C were transferred to the oviducts of pseudopregnant females and recollected 94 h later (treatment III). Diameters of morphologically normal blastocysts were measured prior to incubation for 3 h at 37°C in 3H-uridine (14.8 Ci/mmole, International Chemical and Nuclear Corporation), diluted in 0.2 ml BSM II to 5 &i/ml. To insure measurable incorporation of aH-uridine 15 embryos were usually incubated and analysed together. After incubation with the labeled nucleoside, blastocysts were washed 4 times in BSM II and placed in glass culture tubes. To analyse the amount of 3H-uridine incorporated into RNA, cell membranes were ruptured and the RNA released by subjecting the embryos to a series of 10 rapid freeze-thawings. This was accomplished by moving the culture tubes quickly and alternately between liauid nitrogen and a warm water bath at 40°C. Nucleic acids were precipitated by the addition of 2 ml of cold (4°C) 10 % trichloroacetic acid (TCA). To facilitate precipitation of the small amount of labeled embryonic RNA, 1 mg of yeast RNA (Nutritional Biochemical Corporation) in 0.25 ml Tris buffer (nH = 7.0) was added to each tube of embryos. Blanks containing only the yeast RNA, but no embryos, were run with each grouu of samples in order to determine the background. The tubes- were held at 4°C for at least 15 min to insure complete precipitation. The procedure for separating the unincorporated aHuridine and the 3H-uridine labeled RNA was an adaptation of that used by El-Banna & Daniel 131.
Nucleic acid and protein metabolism of embryos Table 1. Incorporation
(2k S.E.) of 3H-thymidine Cultured in vitro
Criteria No. of blastocysts 3H-thymidinelabeled cells ( %) Av. diameter (mm) Total cell number
into DNA
75
by blastocys@
Grown in vivo
Non-stored 0)
Stored first (10
Stored first (III)
Non-stored (continuous) (IV)
53
31
27
48
33 *2a 0.13 +o.o01a 138*8=
27&3 0.12+0.001~ 174f26a
68k2 0.87 +0.04 12911263
61&2 0.66 kO.05 993 + 107
Row means with different superscripts differ significantly (p < 0.05). a ‘Stored’ embryos were held at 10°C for 24 h before culture at 37°C or transfer to recipients for development into blastocysts. ‘Non-stored’ embryos were cultured directly at 37°C or allowed to develop into blastocysts in vivo before being collected (continuous).
The TCA containing unincorporated 3H-uridine and the precipitate containing the labeled RNA were poured onto a Gelman Type E fiberglass filter clamped onto a Millipore filter holder and sidearm filter flask. Slight vacuum was applied and the culture tube was washed 5 times with 1 ml cold (4°C) 5 % TCA and the rinse poured onto the filter. The filter containing the RNA was washed an additional 5 times with I ml of cold 5 % TCA and moved to a scintillation vial. After the filter had dried at WC, 14 ml of toluenebased scintillation fluid was added to each vial and the filter counted for radioactivity. An external standard was used to determine the degree of quenching for each sample.
Protein synthesis Two-cell rabbit embryos were assigned to the 5 treatment groups described for the RNA experiment. After diameters were measured, blastocysts were incubated for 3 h at 37°C with 14C-leucine (240 mCi/ mmole, International Chemical and Nuclear Corporation), diluted in 0.2 ml BSM II to 0.5 &i/ml and 9.79 mCi/mmole leucine. To insure measurable counts, IO blastocysts were usually incubated and analysed together. Following incubation they were washed with BSM II containing cold leucine, ruptured by 10 rapid freeze-thawings and the proteins precipitated by addition of 2 ml cold (4°C) 10% TCA. Samples were allowed to sit for 15 min at 4°C to insure thorough precipitation. The precipitate containing proteins and nucleic acids was collected on a filter as described for the isolation of RNA. Two additional steps were employed to gain further separation [3]. The filter was rinsed with a final 10 ml of hot (SO-90°C) 5 % TCA to remove nucleic acids and tRNA bound W-leucine. The filters were also rinsed with 5 ml of cold (4°C) 70% ethanol to facilitate drying. Determination of radioactivity was by liquid scintillation counting as described previously for RNA.
RESULTS DNA synthesis Table 1 summarizes the results obtained with embryos incubated with 3H-thymidine. The unequal numbers of embryos per treatment resulted from embryo death which occurred during storage and the fact that not all preparations were suitable for counting. Embryos stored as two-cell embryos and then cultured to blastocysts (II) were slightly less intensely labeled (27 % versus 33 %) than non-stored embryos (I) which were cultured to blastocysts (p < 0.05). Other differences between thesetwo treatments were not significant (table 1). Embryos stored at 10°C as two-cells but allowed to develop subsequently in normally ovulated does (III) showed a significantly greater proportion of cells labeled, had a greater blastocyst diameter and contained more cells than embryos which had developed continuously in superovulated females (IV) (p < 0.05). The greatest differences were not seenbetween stored and non-stored embryos, but rather between embryos grown in vitro or in vivo. Embryos which developed in vitro had a lower proportion of labeled cells, a Exprl
Cell Res 90 (1975)
16 Anderson and Foote Table 2. Incorporation
(X& S.E.) of 3H-uridine into RNA by blastocy.@ Cultured in vitro
Criteria No. blastocysts Av. diameter (mm) Cpm/embryo Cpm/mm2 of embryo area
Grown in vivo
Non-stored (1)
Stored (11)
Non-stored (III)
Stored (IV
Continuous (VI
204 0.15 kO.004 149& lga
107 0.15+0.009 106i26’
145 0.97 + 0.03 1 878 + 309
161 0.82 k 0.07 1555+330
335 1.30+0.04 145Ok249
625 * 8ga
784&156=
511 k72
Row means with different superscripts differ significantly (p < 0.05). a See table 1.
smaller average blastocyst diameter and fewer total cells (~~0.005) than blastocysts which developed in the female tract.
RNA synthesis Table 2 summarizes the incorporation rates of 3H-uridine by blastocysts. Storage prior to culture in vitro (I vs II) did not significantly alter the rate of incorporation of 3H-uridine into RNA. Since blastocysts from the two treatments had the same average diameter, no adjustment was made for differences in blastocyst size. On the other hand, in order to compare 3H-uridine incorporation at the cellular level into blastocysts of different sizes grown in vivo, corrections were made for differences in blastocyst size (table 2). This correction was based upon the assumptions that at this stage of blastocyst expansion, individual cell size is essentially uniform and constant and precursor pool sizesare the samefor embryos from thesethree treatments. With these assumptions, expression on a unit area basis is also an expression on the basis of a nearly equal number of cells. Stored embryos allowed to develop in vivo (IV) incorporated less total 3H-uridine than did non-stored embryos which developed in vivo (III) (~~0.05). However, after correcting for differences in blastocyst size and number of Exptl
Cell Res 90 (1975)
cells, there was no difference in the amount of 3H-uridine incorporated into RNA per cell (p > 0.05). Embryos which developed into blastocysts in the superovulated females (V) incorporated less 3H-uridine on a per-cell basis than did stored embryos which developed in vivo (IV). Overall, blastocysts which developed in vivo (III, IV and V) incorporated 10-20 times as much 3H-uridine as those which developed in vitro (I, II) (~~0.005). Since endogenous precursor pools of blastocysts grown in vitro were probably different from those grown in vivo, it was not reasonable to make size adjustments for direct comparison at the cellular level.
Protein synthesis Table 3 showsthe relative rates of 14C-leucine incorporation for blastocysts from the five treatment groups described for RNA. No effect of storage at 10°C was observed upon rates of incorporation when embryos were cultured to blastocysts (I and II). While there appeared to be a difference among the embryos grown in various ways in vivo (III, IV and V), these differences disappeared when rates of incorporation were expressed as cpm/mm2 of embryo area to correct for differences in blastocyst size (p >0.05). Blasto-
Nucleic acid and protein metabolism of embryos Table 3. Incorporation
(X f- S. E.) of W-leucine Cultured in vitro
Criteria No. blastocysts Av. diameter (mm) Cpmlembryo Cpm/mm2 of embryos area
17
into protein by blastocysts” Grown in vivo
Non-stored (1)
Stored (11)
Non-stored mu
Stored (IV)
Continuous 09
97 0.13~0.003 7i1.2a
35 0.13 +0.005 8 + 2.8a
85 1.13io.07 350&48
77 0.84 kO.05 199*31
93 0.92 5 0.05 230+29
-
-
91 * 13.0
8728.8
8456.2
ROW means with different superscripts differ significantly (p
Experimental
EFFECTS
OF LOW
ACID
AND
Cell Research 90 (1975) 73-78
TEMPERATURE
PROTEIN
SYNTHESIS
G. B. ANDERSON1 Department
of Animal
Science,
UPON
SUBSEQUENT
OF RABBIT
NUCLEIC
EMBRYOS
and R. H. FOOTE
and Division of Biological Ithaca, N. Y. 14850, USA
Sciences,
Cornell
University,
SUMMARY Two-cell rabbit embryos, which were maintained without cleavage for 24 h at lO”C, and then allowed to develop into blastocysts in vivo or in a completely defined medium at 37”C, incorporated aH-uridine into RNA and WXeucine into protein at rates comparable to blastocysts not previously exposed to 10°C temperature. Thus, temporary suppression of development did not impair nucleic acid and protein synthesis by embryos developing into blastocysts. Blastocysts which developed in vivo incorporated more 3H-thymidine into DNA, 3H-uridine into RNA and 14C-leucine into protein than did blastocysts which developed in vitro. These differences probably reflect not only differences in blastocyst size and number of cells, but also differences in endogenous precursor pools.
Despite widespread manipulation of mammalian embryos in vitro little is known about effects of cooling embryos on subsequent metabolism. Recent studies showed that temporary inhibition of development of rabbit embryos by holding at 10°C for 24 h had no effect upon subsequent pyruvate or glucose utilization when the embryos were rewarmed to 37°C [l]. These findings suggested that embryo death following low temperature stress was not due to an induced inability of the embryo to obtain energy for development. The experiments reported in this paper were designed to determine the rates of incorporation of labeled precursors into DNA, RNA and protein by rabbit blastocysts which had been stored in the two-cell stage at 10°C for 24 h. These were compared with incorporation rates of blastocysts which were not stored at 10°C. 1 Present address: Department of Animal Science, University of California, Davis, Calif. 95616, USA.
MATERIALS
AND METHODS
DNA synthesis Two-cell embryos were collected from superovulated Dutch-Belted rabbits 26 h after LH administration, pooled and then randomly assigned to treatment groups to compare DNA synthesis in vitro and in ;ivo- following- inhibition at 10°C. Embryos were cultured into blastocysts in BSM II, a defined culture medium [4], for 94 h at 37°C (treatment I) or stored for 24 h at 10°C in BSM II plus 4 % Promine-D (w/v). a soybean protein, as previously described [l]. Foll&: ing storage at 10°C embryos were either cultured at 37°C for 94 h (II) or transferred to the oviducts of pseudopregnant females and recollected from the uteri 94 h after transfer (III). Treatment IV consisted of blastocysts collected from the uteri of superovulated females 120 h after LH administration. Since the embryos in treatments II and III did not divide while being held at 10°C in the two-cell stage, blastocysts from all treatments were assumed to represent embryos having undergone 120 h of development. Diameter measurements were determined on morphologically normal blastocysts from each of the treatment groups at the end of the development period. Blastocysts were incubated at 37°C in 0.2 ml BSM II with 3H-methylthymidine (6.7 Ci/mmole, New England Nuclear) added to achieve an activity of 10 ,&i/ml. After incubation in the labeled medium for 1 h, blastocysts were washed 4 times in BSM II to remove unincorporated 3H-thymidine. Following the fourth wash the embryos were moved to a watch Exptl
Cell Res 90 (1975)
74
Anderson and Foote
Fig. I. Autoradiograph of a 120-h rabbit blastocyst (430 x ). Most nuclei which incorporated 3H-thymidine during the incubation period are extensively covered with reduced silver grains. Non-labeled cells were stained bright blue with Harris hematoxylin, but appear gray in the photograph. The arrow indicates a cell in anaphase which was unlabeled, as expected.
glass containing 70% ethanol for 1 to 2 h. The alcohol treatment fixed the embryos, prevented them from synthesizing more DNA and also tended to loosen the intercell matrix so that individual cells saread to form a monolayer when the embryo was squashed on a glass slide. Embryos were then exnosed to a 0.5 % solution of pronase-for 10 to 30 set-followed quickly by a wash with nhvsioloaical saline. Blastocvsts were soread on clean; gelatin-coated glass slides and embryosquashcs ureoared as described bv Sanval & Meyer 16. 71. The slides were coated with Kodak NTB3 nuclear emulsion using a roller technique [S]. They were exposed for 5 days at 4°C followed by development and staining with Harris’ hematoxvlin. All nucl4 were stained blue, but nuclei which incorporated $H-thymidine were labeled with overlying reduced silver grains (fig. 1). The numbers of labeled and unlabeled cells were counted at 100 x magnification and the total number of cells per blastocyst and the percentage of total cells that were labeled were calculated. This latter parameter was used to compare the relative mitotic activity in embryos from the various treatment groups. An analysis of variance for unequal subclasses was used to test treatment differences and Tukey’s hsd test was used to test for differences among treatment means.
RNA synthesis Two-cell embryos were collected as for the DNA studies and assigned to 1 of 4 treatment groups. Treatments are indicated in table 2. Treatments I, II, Exptl
Cell Res 90 (1975)
1V and V correspond to treatments I-IV in the previous experiment (table 1). In addition, collected embryos which were not held at 10°C were transferred to the oviducts of pseudopregnant females and recollected 94 h later (treatment III). Diameters of morphologically normal blastocysts were measured prior to incubation for 3 h at 37°C in 3H-uridine (14.8 Ci/mmole, International Chemical and Nuclear Corporation), diluted in 0.2 ml BSM II to 5 &i/ml. To insure measurable incorporation of aH-uridine 15 embryos were usually incubated and analysed together. After incubation with the labeled nucleoside, blastocysts were washed 4 times in BSM II and placed in glass culture tubes. To analyse the amount of 3H-uridine incorporated into RNA, cell membranes were ruptured and the RNA released by subjecting the embryos to a series of 10 rapid freeze-thawings. This was accomplished by moving the culture tubes quickly and alternately between liauid nitrogen and a warm water bath at 40°C. Nucleic acids were precipitated by the addition of 2 ml of cold (4°C) 10 % trichloroacetic acid (TCA). To facilitate precipitation of the small amount of labeled embryonic RNA, 1 mg of yeast RNA (Nutritional Biochemical Corporation) in 0.25 ml Tris buffer (nH = 7.0) was added to each tube of embryos. Blanks containing only the yeast RNA, but no embryos, were run with each grouu of samples in order to determine the background. The tubes- were held at 4°C for at least 15 min to insure complete precipitation. The procedure for separating the unincorporated aHuridine and the 3H-uridine labeled RNA was an adaptation of that used by El-Banna & Daniel 131.
Nucleic acid and protein metabolism of embryos Table 1. Incorporation
(2k S.E.) of 3H-thymidine Cultured in vitro
Criteria No. of blastocysts 3H-thymidinelabeled cells ( %) Av. diameter (mm) Total cell number
into DNA
75
by blastocys@
Grown in vivo
Non-stored 0)
Stored first (10
Stored first (III)
Non-stored (continuous) (IV)
53
31
27
48
33 *2a 0.13 +o.o01a 138*8=
27&3 0.12+0.001~ 174f26a
68k2 0.87 +0.04 12911263
61&2 0.66 kO.05 993 + 107
Row means with different superscripts differ significantly (p < 0.05). a ‘Stored’ embryos were held at 10°C for 24 h before culture at 37°C or transfer to recipients for development into blastocysts. ‘Non-stored’ embryos were cultured directly at 37°C or allowed to develop into blastocysts in vivo before being collected (continuous).
The TCA containing unincorporated 3H-uridine and the precipitate containing the labeled RNA were poured onto a Gelman Type E fiberglass filter clamped onto a Millipore filter holder and sidearm filter flask. Slight vacuum was applied and the culture tube was washed 5 times with 1 ml cold (4°C) 5 % TCA and the rinse poured onto the filter. The filter containing the RNA was washed an additional 5 times with I ml of cold 5 % TCA and moved to a scintillation vial. After the filter had dried at WC, 14 ml of toluenebased scintillation fluid was added to each vial and the filter counted for radioactivity. An external standard was used to determine the degree of quenching for each sample.
Protein synthesis Two-cell rabbit embryos were assigned to the 5 treatment groups described for the RNA experiment. After diameters were measured, blastocysts were incubated for 3 h at 37°C with 14C-leucine (240 mCi/ mmole, International Chemical and Nuclear Corporation), diluted in 0.2 ml BSM II to 0.5 &i/ml and 9.79 mCi/mmole leucine. To insure measurable counts, IO blastocysts were usually incubated and analysed together. Following incubation they were washed with BSM II containing cold leucine, ruptured by 10 rapid freeze-thawings and the proteins precipitated by addition of 2 ml cold (4°C) 10% TCA. Samples were allowed to sit for 15 min at 4°C to insure thorough precipitation. The precipitate containing proteins and nucleic acids was collected on a filter as described for the isolation of RNA. Two additional steps were employed to gain further separation [3]. The filter was rinsed with a final 10 ml of hot (SO-90°C) 5 % TCA to remove nucleic acids and tRNA bound W-leucine. The filters were also rinsed with 5 ml of cold (4°C) 70% ethanol to facilitate drying. Determination of radioactivity was by liquid scintillation counting as described previously for RNA.
RESULTS DNA synthesis Table 1 summarizes the results obtained with embryos incubated with 3H-thymidine. The unequal numbers of embryos per treatment resulted from embryo death which occurred during storage and the fact that not all preparations were suitable for counting. Embryos stored as two-cell embryos and then cultured to blastocysts (II) were slightly less intensely labeled (27 % versus 33 %) than non-stored embryos (I) which were cultured to blastocysts (p < 0.05). Other differences between thesetwo treatments were not significant (table 1). Embryos stored at 10°C as two-cells but allowed to develop subsequently in normally ovulated does (III) showed a significantly greater proportion of cells labeled, had a greater blastocyst diameter and contained more cells than embryos which had developed continuously in superovulated females (IV) (p < 0.05). The greatest differences were not seenbetween stored and non-stored embryos, but rather between embryos grown in vitro or in vivo. Embryos which developed in vitro had a lower proportion of labeled cells, a Exprl
Cell Res 90 (1975)
16 Anderson and Foote Table 2. Incorporation
(X& S.E.) of 3H-uridine into RNA by blastocy.@ Cultured in vitro
Criteria No. blastocysts Av. diameter (mm) Cpm/embryo Cpm/mm2 of embryo area
Grown in vivo
Non-stored (1)
Stored (11)
Non-stored (III)
Stored (IV
Continuous (VI
204 0.15 kO.004 149& lga
107 0.15+0.009 106i26’
145 0.97 + 0.03 1 878 + 309
161 0.82 k 0.07 1555+330
335 1.30+0.04 145Ok249
625 * 8ga
784&156=
511 k72
Row means with different superscripts differ significantly (p < 0.05). a See table 1.
smaller average blastocyst diameter and fewer total cells (~~0.005) than blastocysts which developed in the female tract.
RNA synthesis Table 2 summarizes the incorporation rates of 3H-uridine by blastocysts. Storage prior to culture in vitro (I vs II) did not significantly alter the rate of incorporation of 3H-uridine into RNA. Since blastocysts from the two treatments had the same average diameter, no adjustment was made for differences in blastocyst size. On the other hand, in order to compare 3H-uridine incorporation at the cellular level into blastocysts of different sizes grown in vivo, corrections were made for differences in blastocyst size (table 2). This correction was based upon the assumptions that at this stage of blastocyst expansion, individual cell size is essentially uniform and constant and precursor pool sizesare the samefor embryos from thesethree treatments. With these assumptions, expression on a unit area basis is also an expression on the basis of a nearly equal number of cells. Stored embryos allowed to develop in vivo (IV) incorporated less total 3H-uridine than did non-stored embryos which developed in vivo (III) (~~0.05). However, after correcting for differences in blastocyst size and number of Exptl
Cell Res 90 (1975)
cells, there was no difference in the amount of 3H-uridine incorporated into RNA per cell (p > 0.05). Embryos which developed into blastocysts in the superovulated females (V) incorporated less 3H-uridine on a per-cell basis than did stored embryos which developed in vivo (IV). Overall, blastocysts which developed in vivo (III, IV and V) incorporated 10-20 times as much 3H-uridine as those which developed in vitro (I, II) (~~0.005). Since endogenous precursor pools of blastocysts grown in vitro were probably different from those grown in vivo, it was not reasonable to make size adjustments for direct comparison at the cellular level.
Protein synthesis Table 3 showsthe relative rates of 14C-leucine incorporation for blastocysts from the five treatment groups described for RNA. No effect of storage at 10°C was observed upon rates of incorporation when embryos were cultured to blastocysts (I and II). While there appeared to be a difference among the embryos grown in various ways in vivo (III, IV and V), these differences disappeared when rates of incorporation were expressed as cpm/mm2 of embryo area to correct for differences in blastocyst size (p >0.05). Blasto-
Nucleic acid and protein metabolism of embryos Table 3. Incorporation
(X f- S. E.) of W-leucine Cultured in vitro
Criteria No. blastocysts Av. diameter (mm) Cpmlembryo Cpm/mm2 of embryos area
17
into protein by blastocysts” Grown in vivo
Non-stored (1)
Stored (11)
Non-stored mu
Stored (IV)
Continuous 09
97 0.13~0.003 7i1.2a
35 0.13 +0.005 8 + 2.8a
85 1.13io.07 350&48
77 0.84 kO.05 199*31
93 0.92 5 0.05 230+29
-
-
91 * 13.0
8728.8
8456.2
ROW means with different superscripts differ significantly (p
