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Factors influencing the in vitro hatching of mouse blastocysts
Animal Reproduction Science, 1 (1978) 181--188
181
© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
FACTORS INFLUENCING THE IN VITRO HATCHING OF MOUSE BLASTOCYSTS
RAYMOND W. WRIGHT, JR.*, J.G. WATSON** and STERLING CHAYKIN**
*Department of Animal Sciences, Washington State University, Pullman, Wash. 99163 (U.S.A.) **Department of Biochemistry and Biophysics, University of California, Davis, Calif. 95616 (U.S.A.) Scientific Paper No. 4768. College of Agriculture Research Center, Washington State University, Pullman. Project 0313. (Received 4 August 1977)
ABSTRACT Wright, R.W., Jr., Watson, J.G. and Chaykin, S., 1978. Factors influencing the in vitro hatching of mouse blastocysts. Anim. Reprod. Sc£ 1: 181--188. The influence of bovine serum albumin (BSA) concentration on embryo hatching and the number of embryos cultured per drop of culture medium was examined in F1 (C57BL/6J x DBA/2J), C3HeB/FeJ strain and Line E mice. Embryos collected from F1 and Line E mice exhibited uniform hatching rates at BSA concentrations between 1 and 10 mg/ml, and embryo numbers ranging from 1 to 10 per 3 al of culture medium. The hatching of C3HeB/FeJ blastocysts was greater at the higher concentrations of BSA and higher embryo densities. When the C3HeB/FeJ embryos were grown at high densities until morula and then cultured singly in fresh media they hatched at a low rate. However, when allowed to develop until the blastocyst stage before replotting, the embryos hatched at a high rate. C3HeB/FeJ embryos cultured singly until morula and then placed in groups of 10 hatched at a high rate. Single C3HeB/FeJ embryos, cultured in medium conditioned by the prior presence of embryos at high densities, hatched at a slightly higher frequency than those cultured in fresh medium. There was no tendency of embryos developing from the two-cell to the eight-cell stages to hatch when cultured in the presence of high densities of hatching blastocysts.
INTRODUCTION Bovine s e r u m a l b u m i n ( B S A ) is o f t e n i n c l u d e d as a c o n s t i t u e n t o f m e d i a u s e d in t h e c u l t u r e o f p r e i m p l a n t a t i o n m a m m a l i a n e m b r y o s b e c a u s e it has been shown to have a beneficial effect on d e v e l o p m e n t and b l a s t o c y s t escape ( C h o l e w a a n d W h i t t e n , ! 9 7 0 ) . Its p r e s e n c e , h o w e v e r , is n o t o b l i g a t o r y f o r n o r m a l d e v e l o p m e n t . C h o l e w a a n d W h i t t e n ( 1 9 7 0 ) also d e m o n s t r a t e d t h a t p r e i m p l a n t a t i o n m o u s e e m b r y o s c o u l d d e v e l o p i n t o b l a s t o c y s t s in v i t r o in t h e a b s e n c e of an e x o g e n o u s f i x e d n i t r o g e n s o u r c e w h e n a high m o l e c u l a r w e i g h t c o l l o i d , such ~ p e l y v i n y l p y r e l i d o n e , was s u b s t i t u t e d f o r B S A in t h e
182
ctflture medium. They at~ributed the beneficial albumin and amino acid effect to, ~be chelation of heavy metals and membrane stabilization as opposed to a nutritional phenomenon. Additional evidence that the BSA effect may no~ be entirely the result of the release of its constituent amino acids comes from the work of Brinster {1971), in which it was shown that albumin does n o t compete with the uptake of labelled amino acids by mouse blastocysts. Spindle and Pedersen {1973), however, have demonstrated that hatching can be facilitated by certain essential amino acids, while other amino acids are without effect. These results have been difficult to relate with earlier data because of the rate of blastocysts hatching (67%) and the lack of definition introduced into the culture medium by the inclusion of 1% fetal calf serum, because the density of embryos per volume of culture medium was not specified, and the efficacy of each of the 12 amino acids examined was evaluated by omission from, rather than inclusion in, the medium. In light of the apparent complexity of the response of preimplantation embryos to proteins and amino acids the entire p h e n o m e n o n must be further characterized. This report presents data which bear on the interaction of strain, e m b r y o density in the culture medium, and BSA concentration as factors regulating hatchability of mouse blastocysts. MATERIALS AND METHODS
F1 (C57BL/6J × DBA/2J), C3HeB/FeJ, and Line E {Bradford, 1969) mice (6 to 10 week old) were raised in our own colony or purchased from the Jackson Laboratory, Bar Harbor, Maine. Whitten's medium (Whitten and Biggers, 1968) for mouse e m b r y o culture was modified to contain 1.0, 2.5, 5.0, 7.5, or 10 mg/ml BSA {Sigma, crystalline). Virgin female mice were superovulated and artificially inseminated with spermatozoa from C3HeB/ FeJ males as described by Leckie et al. (1973). Embryos at the two-cell stage were obtained 36 h after insemination by flushing the oviducts with Whitten's medium 1 mg/ml BSA. Normal appearing embryos were pooled, washed three times with the particular medium to be used for culture and placed randomly among the various treatment groups. Incubations were carried out at 37°C in a humidity controlled water jacketed incubator with a 5% CO2 in air atmosphere. Embryos were maintained in microdrops (3 ~1) of medium, under paraffin oil, in a plastic culture dish (Brinster, 1963). For experimental purposes hatching was defined as the complete escape of a blastocyst from the zona pellucida. Only embryos which had successfully completed in vitro development from the two-cell to the blastocyst stage were used in these experiments. Hatching in the various culture media was compared within and among the treatment groups. Hatching responses were evaluated using the Student's t-test.
183
RESULTS
Development and the hatching process in both the F1 and Line E mice we~.e unaffected by either varying the BSA concentration in the medium from ?: to ~.0 mg/ml, ~r increasing t!~e number of embryos cultured from 1 tv 19 per microdrop (Tables I and II). Under all the conditions tested in this experiment, hatching rates were at or above 90%. Within the limits of the experimental conditions there was no effect on the numbers of embryos which developed from the two-cell stage to the blastocyst stage, and no change in the time it t o o k for individual embryos to develop from the twoccll to the blastula stage. TABLE I Effect of increasing concentrations of bovine serum albumin and the number of embryos cultured per drop of medium on hatching of Fl[C57BL/6J × DBA/2J] x C3HeB/FeJ blastocysts Embryos hatching per replicate (mean +- S E ) I BSA (mg/ml)
One embryo per drop
Three embryos per drop
10 embryos per drop
0.0 1.0 2.5 5.0 7.5 10.0
0.5±0.1 9.020.4 9.3±0.2 9.620.2 9.3±0.9 9.8±0.1
0.7±0.1 9.120.4 9.320.2 9.420.5 9.2±0.2 9.320.4
1.0±0.2 9.3~0.3 9.3±0.2 10.0±0.0 9.5~0.1 9.820.2
Five replicates per treatment with ten embryos per replicate.
TABLE II Effect of increasing concentrations of bovine serum albumin and the number of embryos cultured per drop of medium on hatching of Line E x C3HeB/FeJ blastocysts Embryos hatching per replicate (mean + SE) 1 BSA (mg/ml)
One embryo per drop
Three embryos per drop
10 embryos per drop
0.0 1.0 2.5 5.0 7.5 10.0
0.1±0.0 9.820.2 9.3±0.1 9.5±0.4 9.020.5 9.6±0.2
0.4±0.1 9.1±0.1 10.0±0.0 9.4±0.3 9.020.2 9.020.4
0.9±0.2 10.0±0.0 9.4±0.1 9.8±0.1 9.6±0.5 9.7±0.2
i Five replicates per treatment with 10 embryos per replicate.
184
By contrast, hatching of C3HeB/FeJ embryos was dependent on embryo density and BSA concentration (1 to 10 mg/ml) in the culture medium (Fig.l). A six-fold increase in the hatchability of singly cultured embryos was observed when the BSA concentration was raised from 1 to 2.5 mg/ml. There was a comparable increase in hatchability (seven-fold) when the embryo density was increased from 1 to 10 embryos per drop of medium at a constant BSA concentration of I mg/ml. Once this initially large increase in hatchability was induced, either by BSA or high embryo density, the responses to further increases in the concentration of BSA were linear and independent of embryo density. Over the mid-range of BSA concentrations there was a constant embryo--density dependent, difference in hatchability, with 0.5 more embryos hatching at a density of three embryos per drop as compared to one embryo per drop, and 1.3 more embryos hatching at a density of 10 compared to one. In a separate experiment it was found that there was a linear increase in the percentage of embryos hatching up to a density of five embryos per microdrop of medium containing I mg BSA/ml, and that the response of hatching to increasing embryo density quickly declined so that the percentage of embryos which hatched at densities of 10 and 30 embryos per microdrop was the same (Fig.2). 100
80
x..~ .~'~" ~ J e / ~ .*+ ~'+ ~'+'~l~ j ~ / ' 0 ~ ~
X GI IAI
60
~'~' !
+
~
°/
/
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40
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20
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4 BSA
6 ( rng/ml
8
10
)
Fig.1. Effect o f increasing concentrations of BSA and numbers of embryos cultured per drop of m e d i u m on the hatching o f C3HeB/FeJ × C3HeB/FeJ blastocysts. Each point represents the average o f five replicates o f 10 embryos per replicate..-.× . . . . 10 embryos per drop;
0
= three embryos per drop;
0
= one e m b r y o per drop.
185
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60 -O . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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iI /i /0 i! I0
I 5
I 10
I 15
i 20
I 25
i 30
EMBRYOS PER DROP
Fig.2. Effect of increasing densities of embryos on the hatching percentage of C3HeB/FeJ x C3HeB/FeJ blastocysts. The standard medium containing 1 mg/rnl of BSA was used. Each point represents the average of five replicates of 10 embryos per replicate, all with a SEM of less than two percent.
While n o t directly related to the issue of the hatchability of blastocysts, it was noted that there was a 12-h delay in the development of C3HeB/FeJ X C3HeB/FeJ embryos at low BSA concentrations and low e m b r y o numbers. In the absence of BSA, embryos of all three types developed poorly, with few two.cell embryos achieving the blastocyst stage. Of those that appeared to be normal at the blastula stage, less than 10% hatched. These results led to a re-examination of the BSA dependence of the hatchability of Fl X C3HeB/FeJ embryos. Although the data are not presented here, the results for the F1 X C3HeB/FeJ embryos were similar to those for the C3HeB/FeJ X C3HeB/FeJ embryos, except the former embryos did not exhibit reduced hatching rates until the BSA levels were lowered to one-tenth the concentration, which caused hatching rates in the latter e m b r y o type to be reduced. The basis of the e m b r y o density enhancement of hatching in the C3HeB/ FeJ X C3HeB/FeJ embryos was subjected to further examination (Table III}. The e x p e r i m e n b l ~ppro,~ch was to evaluate the developmental stages at which embryos could be cultured singly without deleterious effects on hatchability. A culture medium containing BSA at a concentration of I mg/ml was used i.~ order to accentuate the dependency of hatchability on e m b r y o numbers. Groups ! and 2 represent the extremes of the e m b r y o density effect on h_~.tcl~ability. Embryos cultured singly hatched at a 14.7% rate. In g~o~ps of 10 the rate was 57.6%. Embryos cultured through the morula s~age in greups of 10 and then singly, hatched at a rate comparable to that for embryos cultured singly ~;hroughcu:~ (group 3), whereas those cultured through the blastocyst stage before being cultured singly hatched at the same rate as embryos kept in groups of 10 throughout (group 4). Combining embryos, which had been cultured singly through the morula stage, into groups of 10 led to a high rate of hatching (group 5). Finally, incubating individual two-cell embryos in a microdrop conditioned by both the prior
.
--morula blastocyst morula blastocyst 2-cell
standard standard standard standard standard standard
.
Stage o f change
Medium
Embryos per drop
1 10 10 10 1 10 . .
Final
Initial
Culture c o n d i t i o n s I
n o change n o change 1 1 10 9 1
Embryos per d r o p --fresh fresh fresh no change conditioned
Medium
95 125 43 82 44 323 42
Number of embryos
14 72 8 44 27 190 13
Number hatched
14.7 57.6 18.6 53.7 61.4 58.8 31.0
Percent hatched 3
S t a n d a r d culture m e d i u m c o n t a i n i n g 1 mg BSA/ml. 2 T w o cell e m b r y o s were c u l t u r e d at a d e n s i t y o f 10 per drop. When t h e y had achieved t h e early b l a s t o c y s t stage o n e e m b r y o was r e m o v e d f r o m each d r o p a n d o n e freshly c o l l e c t e d two-cell e m b r y o p u t in its place. The h a t c h i n g rate o f t h e nine original b l a s t o c y s t s is r e c o r d e d u n d e r 6a. All o f the original b l a s t o c y s t s were r e m o v e d f r o m t h e d r o p s 48 h a f t e r the two-cell e m b r y o was a d d e d . The two-cell e m b r y o s h a t c h e d at the e x p e c t e d t i m e at the rate r e c o r d e d u n d e r 6b. 3 Percentages with single or d o u b l e bars do n o t differ significantly; t h o s e w i t h single bars differ f r o m t h o s e w i t h d o u b l e (P < 0.01).
1 2 3 4 5 65 a b
Experimental Group
E f f e c t o f culture c o n d i t i o n s o n t h e h a t c h i n g o f C 3 H e B / F e J × C 3 H e B / F e J b l a s t o c y s t s
T A B L E III
187
development of 10 embryos to the blastocyst stage and the continuing presence of nine of those embryos through hatching (group 6a) did not cause premature hatching of the developing two-cell embryos (hatching before the expanded blastocyst stage); it did lead to a greater hatching rate of the developing two-cell embryos than would have been expected if the medium had not been conditioned (group 6b compared to group 1, P < 0.05). DISCUSSION Hatching of mouse blastocysts in utero appears to be the result of both maternal and embryonic factors (McLaren, 1970; Mintz, 1971). Escape from the zona pellucida is viewed as an embryonic function and lysis of the zona as a uterine function, which can occur either simultaneously with or subsequent to hatching (McLaren, 1967; Orsini and McLaren, 1967; McLaren, 1968). Since hatching in vitro is free of maternal influences, it is characterized by escape of the embryo by rupture of the zona without lytic thinning (Bergstrom, 1972). The physical force generated by the alternating expansion and contraction of the blastocyst, over a period of many hours, is apparently responsible for tearing a hole in the zona through which the blastocyst ultimately escapes (Cole, 1967). In the context of the physiological events in which the blastocyst must engage in order to effect its release from the zona, it was not surprising to find a complex of factors having implications upon the hatching process. While the specific factors are still obscure and some will undoubtedly prove to be second order effects related to the general physiological well being of the blastocyst, strain, embryo and media dependent aspects are clearly involved in the success of blastocyst hatching in vitro. These are exemplified by the differences in the BSA dependence of development and hatching in the three embryo types used in this study. All require BSA, but the C3HeB/FeJ × C3HeB/FeJ embryo is the most fastidious. In the latter case the BSA effect appears to have two facets (Fig.l), one which can be compensated for by increased embryo density in the medium, and a second which appears to be indepe1~dent of emb~,e density. No further augmentation in hatching of singly cultured embryos was achieved by increasing BSA levels beyond 10 mg/ ml (Fig.2), but increased embryo density at 10 mg BSA per ml did increase hatchabili~;y. Thus, it can be concKlded that there is also an embryo related BSA concentration-independent factor operating. The nature of the embryo effect is partially illuminated by the experiments en the Liming cf the expression of the density effect (Table III). It appears that high embryo density is most crucial during develepment from the morula to the expanded blastocyst stage. It now remains to determine the specific nature of the several BSA and embryo dependent effects on hatching, be they nutritional (amino acids), osmotic, inhibitor removal, membrane stabilizing, hormonal (BSA bound hormone(s)), or enzymatic.
188
REFERENCES Bergstrom, S., 1972. Shedding of the zona pellucida in normal pregnancy and various hormonal states in the mouse: A scanning electron microscope study. Z. Anat. Entwicklungsgesch., 137: 143--167. Bradford, G.E., 1969. Genetic control of ovulation rate and embryo survival in mice. I. Response to selection. Genetics, 61: 905--921. Brinster, R.L., 1963. A method for in vitro cultivation of mouse ova from two-cell to blastocyst. Exp. Cell Res., 32: 205--208. Brinster, R.L., 1971. Uptake and incorporation of amino acids by the preimplantation mouse embryo. J. Reprod. Fertil., 27: 329--338. Cholewa, J.A. and Whitten, W.K., 1970. Development of two-cell mouse embryos in the absence of a fixed-nitrogen source. J. Reprod. Fertil., 22: 553--555. Cole, R.J., 1967. Cinemicrographic observations on the trophoblast and zona pellucida of the mouse blastocyst. J. Embryoh Exp. Morphol., 17: 481--490. Leckie, P.A., Watson, J.G. and Chaykin, S., 1973. An improved method for the artificial insemination of the mouse (Mus musculus). Biol. Reprod., 9: 420--424, McLaren, A., 1967. Delayed loss of the zona pellucida from blastocysts of suckling mice. J. Repro& Fertil., 14: 159--162. McLaren, A., 1968. A study of blastocysts during delay and subsequent implantation in lactating mice. J. Endocrinol., 42: 453--463. McLaren, A., 1970. The fate of the zona pellucida in mice. J. Embryoh Exp. Morphoh, 23: 1--19. Mintz, B., 1971. Control of embryo implantation and survival. Adv. Biosci., 6: 317--342. Orsini, M.W. and McLaren, A., 1967. Loss of the zona pellucida in mice, and the effect of tubal ligation and ovariectomy. J. Reprod. Fertil., 13: 484--499. Spindle, A.I. and Pedersen, R.A., 1973. Hatching, attachment, and outgrowth of mouse blastocysts in vitro: fixed nitrogen requirements. J. Exp. Zooh, 186: 305--318. Whitten, W.K. and Biggers, J.D., 1968. Complete development in vitro of the pre-implantation stages of the mouse in a simple chemically defined medium. J. Reprod. Fertih, 17: 399--401.
181
© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
FACTORS INFLUENCING THE IN VITRO HATCHING OF MOUSE BLASTOCYSTS
RAYMOND W. WRIGHT, JR.*, J.G. WATSON** and STERLING CHAYKIN**
*Department of Animal Sciences, Washington State University, Pullman, Wash. 99163 (U.S.A.) **Department of Biochemistry and Biophysics, University of California, Davis, Calif. 95616 (U.S.A.) Scientific Paper No. 4768. College of Agriculture Research Center, Washington State University, Pullman. Project 0313. (Received 4 August 1977)
ABSTRACT Wright, R.W., Jr., Watson, J.G. and Chaykin, S., 1978. Factors influencing the in vitro hatching of mouse blastocysts. Anim. Reprod. Sc£ 1: 181--188. The influence of bovine serum albumin (BSA) concentration on embryo hatching and the number of embryos cultured per drop of culture medium was examined in F1 (C57BL/6J x DBA/2J), C3HeB/FeJ strain and Line E mice. Embryos collected from F1 and Line E mice exhibited uniform hatching rates at BSA concentrations between 1 and 10 mg/ml, and embryo numbers ranging from 1 to 10 per 3 al of culture medium. The hatching of C3HeB/FeJ blastocysts was greater at the higher concentrations of BSA and higher embryo densities. When the C3HeB/FeJ embryos were grown at high densities until morula and then cultured singly in fresh media they hatched at a low rate. However, when allowed to develop until the blastocyst stage before replotting, the embryos hatched at a high rate. C3HeB/FeJ embryos cultured singly until morula and then placed in groups of 10 hatched at a high rate. Single C3HeB/FeJ embryos, cultured in medium conditioned by the prior presence of embryos at high densities, hatched at a slightly higher frequency than those cultured in fresh medium. There was no tendency of embryos developing from the two-cell to the eight-cell stages to hatch when cultured in the presence of high densities of hatching blastocysts.
INTRODUCTION Bovine s e r u m a l b u m i n ( B S A ) is o f t e n i n c l u d e d as a c o n s t i t u e n t o f m e d i a u s e d in t h e c u l t u r e o f p r e i m p l a n t a t i o n m a m m a l i a n e m b r y o s b e c a u s e it has been shown to have a beneficial effect on d e v e l o p m e n t and b l a s t o c y s t escape ( C h o l e w a a n d W h i t t e n , ! 9 7 0 ) . Its p r e s e n c e , h o w e v e r , is n o t o b l i g a t o r y f o r n o r m a l d e v e l o p m e n t . C h o l e w a a n d W h i t t e n ( 1 9 7 0 ) also d e m o n s t r a t e d t h a t p r e i m p l a n t a t i o n m o u s e e m b r y o s c o u l d d e v e l o p i n t o b l a s t o c y s t s in v i t r o in t h e a b s e n c e of an e x o g e n o u s f i x e d n i t r o g e n s o u r c e w h e n a high m o l e c u l a r w e i g h t c o l l o i d , such ~ p e l y v i n y l p y r e l i d o n e , was s u b s t i t u t e d f o r B S A in t h e
182
ctflture medium. They at~ributed the beneficial albumin and amino acid effect to, ~be chelation of heavy metals and membrane stabilization as opposed to a nutritional phenomenon. Additional evidence that the BSA effect may no~ be entirely the result of the release of its constituent amino acids comes from the work of Brinster {1971), in which it was shown that albumin does n o t compete with the uptake of labelled amino acids by mouse blastocysts. Spindle and Pedersen {1973), however, have demonstrated that hatching can be facilitated by certain essential amino acids, while other amino acids are without effect. These results have been difficult to relate with earlier data because of the rate of blastocysts hatching (67%) and the lack of definition introduced into the culture medium by the inclusion of 1% fetal calf serum, because the density of embryos per volume of culture medium was not specified, and the efficacy of each of the 12 amino acids examined was evaluated by omission from, rather than inclusion in, the medium. In light of the apparent complexity of the response of preimplantation embryos to proteins and amino acids the entire p h e n o m e n o n must be further characterized. This report presents data which bear on the interaction of strain, e m b r y o density in the culture medium, and BSA concentration as factors regulating hatchability of mouse blastocysts. MATERIALS AND METHODS
F1 (C57BL/6J × DBA/2J), C3HeB/FeJ, and Line E {Bradford, 1969) mice (6 to 10 week old) were raised in our own colony or purchased from the Jackson Laboratory, Bar Harbor, Maine. Whitten's medium (Whitten and Biggers, 1968) for mouse e m b r y o culture was modified to contain 1.0, 2.5, 5.0, 7.5, or 10 mg/ml BSA {Sigma, crystalline). Virgin female mice were superovulated and artificially inseminated with spermatozoa from C3HeB/ FeJ males as described by Leckie et al. (1973). Embryos at the two-cell stage were obtained 36 h after insemination by flushing the oviducts with Whitten's medium 1 mg/ml BSA. Normal appearing embryos were pooled, washed three times with the particular medium to be used for culture and placed randomly among the various treatment groups. Incubations were carried out at 37°C in a humidity controlled water jacketed incubator with a 5% CO2 in air atmosphere. Embryos were maintained in microdrops (3 ~1) of medium, under paraffin oil, in a plastic culture dish (Brinster, 1963). For experimental purposes hatching was defined as the complete escape of a blastocyst from the zona pellucida. Only embryos which had successfully completed in vitro development from the two-cell to the blastocyst stage were used in these experiments. Hatching in the various culture media was compared within and among the treatment groups. Hatching responses were evaluated using the Student's t-test.
183
RESULTS
Development and the hatching process in both the F1 and Line E mice we~.e unaffected by either varying the BSA concentration in the medium from ?: to ~.0 mg/ml, ~r increasing t!~e number of embryos cultured from 1 tv 19 per microdrop (Tables I and II). Under all the conditions tested in this experiment, hatching rates were at or above 90%. Within the limits of the experimental conditions there was no effect on the numbers of embryos which developed from the two-cell stage to the blastocyst stage, and no change in the time it t o o k for individual embryos to develop from the twoccll to the blastula stage. TABLE I Effect of increasing concentrations of bovine serum albumin and the number of embryos cultured per drop of medium on hatching of Fl[C57BL/6J × DBA/2J] x C3HeB/FeJ blastocysts Embryos hatching per replicate (mean +- S E ) I BSA (mg/ml)
One embryo per drop
Three embryos per drop
10 embryos per drop
0.0 1.0 2.5 5.0 7.5 10.0
0.5±0.1 9.020.4 9.3±0.2 9.620.2 9.3±0.9 9.8±0.1
0.7±0.1 9.120.4 9.320.2 9.420.5 9.2±0.2 9.320.4
1.0±0.2 9.3~0.3 9.3±0.2 10.0±0.0 9.5~0.1 9.820.2
Five replicates per treatment with ten embryos per replicate.
TABLE II Effect of increasing concentrations of bovine serum albumin and the number of embryos cultured per drop of medium on hatching of Line E x C3HeB/FeJ blastocysts Embryos hatching per replicate (mean + SE) 1 BSA (mg/ml)
One embryo per drop
Three embryos per drop
10 embryos per drop
0.0 1.0 2.5 5.0 7.5 10.0
0.1±0.0 9.820.2 9.3±0.1 9.5±0.4 9.020.5 9.6±0.2
0.4±0.1 9.1±0.1 10.0±0.0 9.4±0.3 9.020.2 9.020.4
0.9±0.2 10.0±0.0 9.4±0.1 9.8±0.1 9.6±0.5 9.7±0.2
i Five replicates per treatment with 10 embryos per replicate.
184
By contrast, hatching of C3HeB/FeJ embryos was dependent on embryo density and BSA concentration (1 to 10 mg/ml) in the culture medium (Fig.l). A six-fold increase in the hatchability of singly cultured embryos was observed when the BSA concentration was raised from 1 to 2.5 mg/ml. There was a comparable increase in hatchability (seven-fold) when the embryo density was increased from 1 to 10 embryos per drop of medium at a constant BSA concentration of I mg/ml. Once this initially large increase in hatchability was induced, either by BSA or high embryo density, the responses to further increases in the concentration of BSA were linear and independent of embryo density. Over the mid-range of BSA concentrations there was a constant embryo--density dependent, difference in hatchability, with 0.5 more embryos hatching at a density of three embryos per drop as compared to one embryo per drop, and 1.3 more embryos hatching at a density of 10 compared to one. In a separate experiment it was found that there was a linear increase in the percentage of embryos hatching up to a density of five embryos per microdrop of medium containing I mg BSA/ml, and that the response of hatching to increasing embryo density quickly declined so that the percentage of embryos which hatched at densities of 10 and 30 embryos per microdrop was the same (Fig.2). 100
80
x..~ .~'~" ~ J e / ~ .*+ ~'+ ~'+'~l~ j ~ / ' 0 ~ ~
X GI IAI
60
~'~' !
+
~
°/
/
pZ W
40
eL
20
2
4 BSA
6 ( rng/ml
8
10
)
Fig.1. Effect o f increasing concentrations of BSA and numbers of embryos cultured per drop of m e d i u m on the hatching o f C3HeB/FeJ × C3HeB/FeJ blastocysts. Each point represents the average o f five replicates o f 10 embryos per replicate..-.× . . . . 10 embryos per drop;
0
= three embryos per drop;
0
= one e m b r y o per drop.
185
t'~ IM
60 -O . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
50 I-< " r 4O
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O
o
j/
I-- 3 0 Z I.IJ (.) 2 0 n," W ,m 10
/j /
/
iI /i /0 i! I0
I 5
I 10
I 15
i 20
I 25
i 30
EMBRYOS PER DROP
Fig.2. Effect of increasing densities of embryos on the hatching percentage of C3HeB/FeJ x C3HeB/FeJ blastocysts. The standard medium containing 1 mg/rnl of BSA was used. Each point represents the average of five replicates of 10 embryos per replicate, all with a SEM of less than two percent.
While n o t directly related to the issue of the hatchability of blastocysts, it was noted that there was a 12-h delay in the development of C3HeB/FeJ X C3HeB/FeJ embryos at low BSA concentrations and low e m b r y o numbers. In the absence of BSA, embryos of all three types developed poorly, with few two.cell embryos achieving the blastocyst stage. Of those that appeared to be normal at the blastula stage, less than 10% hatched. These results led to a re-examination of the BSA dependence of the hatchability of Fl X C3HeB/FeJ embryos. Although the data are not presented here, the results for the F1 X C3HeB/FeJ embryos were similar to those for the C3HeB/FeJ X C3HeB/FeJ embryos, except the former embryos did not exhibit reduced hatching rates until the BSA levels were lowered to one-tenth the concentration, which caused hatching rates in the latter e m b r y o type to be reduced. The basis of the e m b r y o density enhancement of hatching in the C3HeB/ FeJ X C3HeB/FeJ embryos was subjected to further examination (Table III}. The e x p e r i m e n b l ~ppro,~ch was to evaluate the developmental stages at which embryos could be cultured singly without deleterious effects on hatchability. A culture medium containing BSA at a concentration of I mg/ml was used i.~ order to accentuate the dependency of hatchability on e m b r y o numbers. Groups ! and 2 represent the extremes of the e m b r y o density effect on h_~.tcl~ability. Embryos cultured singly hatched at a 14.7% rate. In g~o~ps of 10 the rate was 57.6%. Embryos cultured through the morula s~age in greups of 10 and then singly, hatched at a rate comparable to that for embryos cultured singly ~;hroughcu:~ (group 3), whereas those cultured through the blastocyst stage before being cultured singly hatched at the same rate as embryos kept in groups of 10 throughout (group 4). Combining embryos, which had been cultured singly through the morula stage, into groups of 10 led to a high rate of hatching (group 5). Finally, incubating individual two-cell embryos in a microdrop conditioned by both the prior
.
--morula blastocyst morula blastocyst 2-cell
standard standard standard standard standard standard
.
Stage o f change
Medium
Embryos per drop
1 10 10 10 1 10 . .
Final
Initial
Culture c o n d i t i o n s I
n o change n o change 1 1 10 9 1
Embryos per d r o p --fresh fresh fresh no change conditioned
Medium
95 125 43 82 44 323 42
Number of embryos
14 72 8 44 27 190 13
Number hatched
14.7 57.6 18.6 53.7 61.4 58.8 31.0
Percent hatched 3
S t a n d a r d culture m e d i u m c o n t a i n i n g 1 mg BSA/ml. 2 T w o cell e m b r y o s were c u l t u r e d at a d e n s i t y o f 10 per drop. When t h e y had achieved t h e early b l a s t o c y s t stage o n e e m b r y o was r e m o v e d f r o m each d r o p a n d o n e freshly c o l l e c t e d two-cell e m b r y o p u t in its place. The h a t c h i n g rate o f t h e nine original b l a s t o c y s t s is r e c o r d e d u n d e r 6a. All o f the original b l a s t o c y s t s were r e m o v e d f r o m t h e d r o p s 48 h a f t e r the two-cell e m b r y o was a d d e d . The two-cell e m b r y o s h a t c h e d at the e x p e c t e d t i m e at the rate r e c o r d e d u n d e r 6b. 3 Percentages with single or d o u b l e bars do n o t differ significantly; t h o s e w i t h single bars differ f r o m t h o s e w i t h d o u b l e (P < 0.01).
1 2 3 4 5 65 a b
Experimental Group
E f f e c t o f culture c o n d i t i o n s o n t h e h a t c h i n g o f C 3 H e B / F e J × C 3 H e B / F e J b l a s t o c y s t s
T A B L E III
187
development of 10 embryos to the blastocyst stage and the continuing presence of nine of those embryos through hatching (group 6a) did not cause premature hatching of the developing two-cell embryos (hatching before the expanded blastocyst stage); it did lead to a greater hatching rate of the developing two-cell embryos than would have been expected if the medium had not been conditioned (group 6b compared to group 1, P < 0.05). DISCUSSION Hatching of mouse blastocysts in utero appears to be the result of both maternal and embryonic factors (McLaren, 1970; Mintz, 1971). Escape from the zona pellucida is viewed as an embryonic function and lysis of the zona as a uterine function, which can occur either simultaneously with or subsequent to hatching (McLaren, 1967; Orsini and McLaren, 1967; McLaren, 1968). Since hatching in vitro is free of maternal influences, it is characterized by escape of the embryo by rupture of the zona without lytic thinning (Bergstrom, 1972). The physical force generated by the alternating expansion and contraction of the blastocyst, over a period of many hours, is apparently responsible for tearing a hole in the zona through which the blastocyst ultimately escapes (Cole, 1967). In the context of the physiological events in which the blastocyst must engage in order to effect its release from the zona, it was not surprising to find a complex of factors having implications upon the hatching process. While the specific factors are still obscure and some will undoubtedly prove to be second order effects related to the general physiological well being of the blastocyst, strain, embryo and media dependent aspects are clearly involved in the success of blastocyst hatching in vitro. These are exemplified by the differences in the BSA dependence of development and hatching in the three embryo types used in this study. All require BSA, but the C3HeB/FeJ × C3HeB/FeJ embryo is the most fastidious. In the latter case the BSA effect appears to have two facets (Fig.l), one which can be compensated for by increased embryo density in the medium, and a second which appears to be indepe1~dent of emb~,e density. No further augmentation in hatching of singly cultured embryos was achieved by increasing BSA levels beyond 10 mg/ ml (Fig.2), but increased embryo density at 10 mg BSA per ml did increase hatchabili~;y. Thus, it can be concKlded that there is also an embryo related BSA concentration-independent factor operating. The nature of the embryo effect is partially illuminated by the experiments en the Liming cf the expression of the density effect (Table III). It appears that high embryo density is most crucial during develepment from the morula to the expanded blastocyst stage. It now remains to determine the specific nature of the several BSA and embryo dependent effects on hatching, be they nutritional (amino acids), osmotic, inhibitor removal, membrane stabilizing, hormonal (BSA bound hormone(s)), or enzymatic.
188
REFERENCES Bergstrom, S., 1972. Shedding of the zona pellucida in normal pregnancy and various hormonal states in the mouse: A scanning electron microscope study. Z. Anat. Entwicklungsgesch., 137: 143--167. Bradford, G.E., 1969. Genetic control of ovulation rate and embryo survival in mice. I. Response to selection. Genetics, 61: 905--921. Brinster, R.L., 1963. A method for in vitro cultivation of mouse ova from two-cell to blastocyst. Exp. Cell Res., 32: 205--208. Brinster, R.L., 1971. Uptake and incorporation of amino acids by the preimplantation mouse embryo. J. Reprod. Fertil., 27: 329--338. Cholewa, J.A. and Whitten, W.K., 1970. Development of two-cell mouse embryos in the absence of a fixed-nitrogen source. J. Reprod. Fertil., 22: 553--555. Cole, R.J., 1967. Cinemicrographic observations on the trophoblast and zona pellucida of the mouse blastocyst. J. Embryoh Exp. Morphol., 17: 481--490. Leckie, P.A., Watson, J.G. and Chaykin, S., 1973. An improved method for the artificial insemination of the mouse (Mus musculus). Biol. Reprod., 9: 420--424, McLaren, A., 1967. Delayed loss of the zona pellucida from blastocysts of suckling mice. J. Repro& Fertil., 14: 159--162. McLaren, A., 1968. A study of blastocysts during delay and subsequent implantation in lactating mice. J. Endocrinol., 42: 453--463. McLaren, A., 1970. The fate of the zona pellucida in mice. J. Embryoh Exp. Morphoh, 23: 1--19. Mintz, B., 1971. Control of embryo implantation and survival. Adv. Biosci., 6: 317--342. Orsini, M.W. and McLaren, A., 1967. Loss of the zona pellucida in mice, and the effect of tubal ligation and ovariectomy. J. Reprod. Fertil., 13: 484--499. Spindle, A.I. and Pedersen, R.A., 1973. Hatching, attachment, and outgrowth of mouse blastocysts in vitro: fixed nitrogen requirements. J. Exp. Zooh, 186: 305--318. Whitten, W.K. and Biggers, J.D., 1968. Complete development in vitro of the pre-implantation stages of the mouse in a simple chemically defined medium. J. Reprod. Fertih, 17: 399--401.