Differential permeability of murine visceral yolk sac to thymidine and to hydroxyurea

DEVELOPMENTAL

45, 74-80 (1975)

BIOLOGY

Differential

Permeability Thymidine

of Murine

Visceral

and to Hydroxyurea

Yolk Sac to l

SUE ANN MILLERS

Department of Environmental, Population Boulder,

and Organismic Biology, Colorado, 80302

University

of Colorado,

AND

MEREDITH Department

of Molecular,

N. RUNNER

Cellular and Developmental Biology, Boulder, Colorado, 80302 Accepted

February

University

of Colorado,

26, 1975

Mouse embryos at the IO-12-somite stage of development were excised from the uterus either with or without the encapsulating visceral yolk sac and were incubated in vitro in 3 x lo-’ M thymidine (methyl-T, 5 &i/ml) for 30 min or in 4 x 10e3 M hydroxyurea for 45 min with [3H]thymidine present for the last 30 min. Radioautograms of nuclei of neural epithelium enabled an estimate of the effectiveness of the barrier imposed by the visceral yolk sac membrane to the passage of thymidine and hydroxyurea. Labeling of nuclei in the neural epithelium showed that the visceral yolk sac caused a 44% decrease in frequency and a 51% decrease in intensity of label. Hydroxyurea inhibited labeling by 15% in frequency and 37% in intensity irrespective of the presence or absence of visceral yolk sac. These results show that hydroxyurea and the presence of visceral yolk sac independently interfered with labeling of the neural epithelium by thymidine and that visceral yolk sac caused a proportionally greater retardation of label than did hydroxyurea. Nuclei of the endodermal epithelium of the intervening yolk sac, following exposure to hydroxyurea, showed a labeling decrease of 44% in frequency and 77% in intensity. The inhibitory effect of hydroxyurea on yolk sac labeling, however, did not alter yolk sac permeability to hydroxyurea. The results indicate that the visceral yolk sac, by offering no detectable barrier to hydroxyurea, permits prompt teratogenic action of hydroxyurea directly upon the embryo and suggest that the visceral yolk sac is a likely candidate to account for reports that the 8.5-day mouse embryo in situ fails to label with radioisotopic thymidine.

within an avascular, diffusion-dependent but differentially permeable barrier (Cole Embryonic development in the mouse and Paul, 1966). Information about perduring the second trimester of pregnancy is meability-transport by the visceral yolk characterized by early organogenesis and sac is important because these early orencapsulation within a yolk sac placenta. ganogenetic stages of the embryo are highly The yolk sac prior to its vascularization, susceptible to induction of malformations days 6.5-8.5, encapsulates the embryo and because the visceral yolk sac during this diffusion stage of existence is the final ’ Supported by grants from the National Institutes barrier for protection from or passage of of Health, National Institute of Dental Research (No. DE-196) and from the National Science Foundation noxious environmental agents. (No. GB 14662). Submitted in partial fulfillment of Functional significance of the visceral the requirements of the degree of Doctor of Philosophy yolk sac has been studied by using agents at the University of Colorado, Boulder, CO. such as trypan blue (Beck et al., 1967) or 2Present address: Department of Anatomy, Harchlorambucil (Kernis, 1971) to study intervard Medical School, Boston, MA 02115; all correspondence to S. A. Miller at this address. ference with embryo-mother exchange and INTRODUCTION

74 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.

MILLER AND RUNNER

to explain teratogenesis. Shepard et al. (1970) have studied metabolism in the 11-somite rat embryo but obtained no data suggestive of the role of the yolk sac during substrate utilization by the implanted mouse embryo. Our in vitro experiments were undertaken in an effort to characterize the visceral yolk sac as a diffusion-transport system. Results have been preliminarily reported (Miller, 1974). Incorporation of thymidine by the neural epithelium was quantitated by radioautography in the presence and absence of visceral yolk sac. Transport by the yolk sac was further characterized by exposing the embryo to hydroxyurea, a teratogen and inhibitor of incorporation of thymidine, in the presence and absence of visceral yolk sac. It was found that the intervening yolk sac retarded incorporation of thymidine but presented only a minor barrier to the inhibitory effects of hydroxyurea. UTERINE

REICHERT’S

ENDOMETRIUM

75

of Yolk Sac

Permeability

METHODS

AND

MATERIALS

Randomly bred female mice were opened on the ninth day (8.5 days of gestation) after a vaginal plug was detected. Embryos were dissected in phosphate-buffered saline and those that had developed to the lo-12-somite stage by 1300 hours were prepared with intact visceral yolk sac (Fig. 1) or the yolk sac was carefully removed. Incubation took place in 35mm petri dishes (Falcon #3001) that were on a rocking tray inside a tissue culture incubator maintained at 37°C in an atmosphere of air. Four embryos per 35mm petri dish were incubated for 30 min with 3 x 10m7M [methyl-3H]thymidine (5 kCi/ml; sp act, 17 Ci/mmole; Schwarz/Mann). Experimental embryos were treated for 45 min with 4 x 10d3 M hydroxyurea (Sigma) with addition of [3H]thymidine for the last 30 min. Embryos were then processed for radioautography according to the tech- not illustrated

MEMBRANE

PARIETAL

ALLANTOIS (IN EXOCOEL) BLOOD ISLAND

YOLK SAC

HEAD PROCESS

VISCERAL AMNION HEART RUDIMENT NEURAL

TAIL

PROCESS

GROOVE POSTERIOR

ANTERIOR

YOLK SAC

GUT

PORTAL

GUT PORTAL THIRD

SOMATOPLEURE

SOMITE YOLK SAC CAVITY PARIETAL YOLK SAC REICHERT’S MEMBRANE TROPHOBLAST UTERINE ENDOMETRIUM ,I

not illustrated

1 mm

FIG. 1. Cutaway illustration of the lo-somite mouse embryo and associated extraembryonic membranes. The yolk sac cavity, parietal yolk sac,Reichert’s membrane,trophoblast and uterine endometrium have not been included in the illustration, but they are listed in order of their respective positions relative to the embrvo.

76

DEVELOPMENTAL BIOLOGY

niques described by Kopriwa and LeBlond (1962). An ocular grid enclosing 15,625 pm2 at 400x was used to circumscribe areas within selected tissues in which all nuclei were counted. Background consisted of approximately 30 grains/15,625 pm* or 0.16 grains/nucleus. This density was insufficient to account for random placement of three grains over the area of a nucleus 10 pm in diameter. Therefore, presence of at least three grains over a nucleus, 19 times background, was adopted as the minimum number of grains per nucleus to score it as “labeled.” The distributions of numbers of grains in these data were skewed toward the left thereby precluding comparison of standard errors of nontransformed data. A labeling index for nuclei of neural epithelium in the open neural groove and for nuclei of the endodermal epithelium of the yolk sac was obtained by calculating the number of labeled nuclei as a fraction of total nuclei within the circumscribed area. Chi-square was used as the statistical test for comparison of frequency of labeling. Probability values of 0.01 or less were considered significant. RESULTS

Permeability of the visceral yolk sac was first determined by labeling with tritiated thymidine in the presence or absence of the encapsulating visceral yolk sac (Fig. 1). The resulting quantitation of radioautograms provided the proportion of nuclei of neural epithelium that were overlain by three or more reduced silver grains, i.e., frequency of labeled nuclei, and the number of reduced silver grains per nucleus, i.e., intensity of label. Permeability of the visceral yolk sac to hydroxyurea was determined by exposing embryos to both hydroxyurea and tritiated thymidine in the presence or absence of the visceral yolk sac and determining frequency and intensity of labeling of nuclei in the neural epithelium. This experimental approach has enabled us to evaluate the effectiveness of labeling

VOLUME 45, 1975

by tritiated thymidine in the presence or absence of the encapsulating visceral yolk sac and with or without addition of hydroxyurea. No label was observed over mitotic figures. The implications of this information are discussed briefly in the next section of this report. Embryos without encapsulating yolk sac exposed to tritiated thymidine alone had 86% of the nuclei of neural epithelium labeled; when the yolk sac was present, significantly fewer (48%) nuclei were labeled (x2 = 347; p < 0.001) (Table 1). The control value of 86%, obtained when the yolk sac was absent, was significantly lowered by hydroxyurea to 73% (x2 = 51.3; P < O.OOl), a reduction of 15.1%. When the intact yolk sac was present, the control value of 48% was lowered to 41% by hydroxyurea, a reduction of 14.6%. Thus, both presence of yolk sac and exposure to hydroxyurea decreased labeling frequency although the reduction due to hydroxyurea with yolk sac present was not statistically significant (x” = 9.3; p > 0.03). Presence of yolk sac decreased the frequency of labeling to a greater extent than did hydroxyurea. Mean number of grains over labeled nuclei of neural epithelium provided an alternate evaluation of the effect of presence or absence of yolk sac encapsulation and presence or absence of hydroxyurea on incorporation of tritiated thymidine. Controls with and without intact yolk sac showed respective means of 7.2 and 14.7 grains per labeled nucleus (Table 1). These two control values were lowered in the presence of hydroxyurea to 4.5 and 9.3, respectively. The presence of yolk sac in both control and hydroxyurea-treated embryos lowered the mean number of grains by 51%; the presence of hydroxyurea in embryos, with and without yolk sacs, decreased the number of grains by 37%. Presence of yolk sac decreased the numbers of grains per labeled nucleus to a greater extent than did hydroxyurea.

MILLER AND RUNNER

TABLE

77

of Yolk Sac

Permeability 1

INCORPORATIONOF THYMIDINE BY NEURAL EPITHELIUM OF THE &~-DAY MOUSE EMBRYO: EFFECT OF YOLK SAC AND HYDROXYUREA~ Control groups

Visceral yolk sac

Absent Labeling index Percent decrease because of Visceral yolk sac Hydroxyurea Mean grains (no.) Percent decrease because of Visceral yolk sac Hydroxyurea Number of observations Nuclei counted Embryos represented Litters represented

0.86 14.7 1,000 6 3

Hydroxyurea-treated

Present

Absent

0.48

groups Present

0.73

44.2 7.2 51.1 -

0.41

15.1 9.3

43.9 14.6 4.5

-

51.7 36.9

36.8 1,000 6 3

1,175 10 6

1,075 8 3

DEmbryos in control groups were incubated in (3H]thymidine (5 &i/ml; sp act, 17 Ci/mmole) for 30 min. Hydroxyurea-treated embryos were incubated for 45 min in 4 x lOma M hydroxyurea and exposed to [3H]thymidine during the last 30 min. A labeling index was recorded for neural epithelium in embryos from each of the four groups. Percent decrease, [1-(control/treated)] x 100, in labeling frequency (labeling index x 100) was calculated to determine the effect of presence of yolk sac and hydroxyurea on labeling by thymidine. Mean numbers of grains were calculated for all nuclei overlain by three or more grains.

The proportional decrease in incorporation of thymidine on account of hydroxyurea, 15.1 and 14.6% in frequency of labeled nuclei and 36.8 and 36.9% in number of grains, was the same regardless of whether the yolk sac was present or not. The visceral yolk sac, by both criteria, offered no barrier to the passage of hydroxyurea. In this study of labeling of neural epithehum, designed to assess permeability of yolk sac to hydroxyurea, the yolk sac barrier was itself exposed to hydroxyurea. The extent to which labeling of nuclei of the yolk sac was inhibited by the presence of hydroxyurea is shown in Table 2. In the absence of hydroxyurea, 82% of yolk sac nuclei were labeled with a mean of 16.6 grains; 46% of the nuclei were labeled with a mean of 3.8 grains in the presence of hydroxyurea. Treatment with hydroxyurea, therefore, reduced the frequency of labeled nuclei by 44% (x” = 145.3; P < 0.001) and reduced the intensity of labeling by 77%. Thus, incorporation of thymidine into nuclei of the encapsulating visceral

TABLE

2

INCORPORATIONOF THYMIDINE BY VISCERAL YOLK SAC NUCLEI: EFFECT OF HYDROXYUREA~ Hydroxyurea

Labeling index Percent decrease Mean grains (no.) Percent decrease Total nuclei counted (no.)

Absent

Present

0.82 16.6 500

0.46 44.0 3.8 77.2 500

“Yolk sac nuclei from control and hydroxyureatreated embryos described for Table 1 provided these data.

yolk sac was shown to be inhibited by hydroxyurea. The possible effect of this inhibition on transport by the yolk sac is discussed below. DISCUSSION

Mouse embryos at 8.5 days of gestation were removed from the uterus by separation between the visceral and parietal yolk sac membranes as was done by Clarkson et al. (1969). Embryos having lo-12 somites

78

DEVELOPMENTAL BIOLOGY

were assigned to one of four groups. Embryos either encapsulated within or freed from visceral yolk sac membrane were labeled with tritiated thymidine; experimental groups were pretreated with hydroxyurea before incubation with hydroxyurea and tritiated thymidine. Radioautograms provided quantitative assessment of frequency and intensity with which silver grains over nuclei were reduced by tritiated thymidine. The visceral yolk sac of the lo-12-somite mouse embryo contains blood islands, but they have not yet organized into the vitelline vessels (Cole and Paul, 1966). At this stage in its development the embryo, therefore, appears to make all exchanges of nutrients and metabolites by means of diffusion and membrane transport. Clarkson et al. (1969) showed that, when cultured for 22 hr within its visceral yolk sac, the 8.5-day embryo doubled its content of nucleic acid and protein. We showed in a separate report (Miller and Runner, submitted) that the 8.5-day mouse embryo is a mosiac of cell populations that incorporate thymidine at different rates and that are differentially inhibited by hydroxyurea. The embryo at the stage used in these experiments is a diffusion-dependent system that lends itself to in uitro experimentation with minimal disruption of the innermost mother-embryo transport barriers. Labeling indices after 30 minutes of exposure to tritiated thymidine were high, as would be expected for rapidly growing embryonic tissue. ‘This observation further supports the suitability of this stage mouse embryo to short term in vitro experimentation. Further discussion of the labeling indices is the subject of a separate report (Miller and Runner, submitted). Barrier Effect of the Visceral Thymidine

Yolk Sac to

The neural epithelium, during this diffusion state of existence, is exposed to amniotic fluid and provides an assay for passage of materials through the visceral yolk sac

VOLUME45, 1975

and across the exocoelom and amniotic space. Furthermore, because of the regular intermitotic migration within the neural epithelium (Sauer, 1935), each nucleus studied had similar access to diffusible substances in the amniotic fluid. These nuclei, therefore, were a convenient indicator of availability of labeled precursor after transport through the intact yolk sac and across the amniotic space. The finding that nuclei of the neural epithelium of the early embryos displayed label from tritiated thymidine (Miller and Runner, submitted) unequvocally showed that the murine visceral yolk sac was permeable to thymidine. This permeability has previously been shown for older embryos when labeled thymidine was administered to pregnant mice (Atlas et al., 1960; Kauffman, 1966, 1968) and to pregnant rats (Scott et al., 1971). The present report of labeling in vitro in the presence and absence of the investing yolk sac membrane showed that, in embryos free of the yolk sac, 86% of the neural nuclei had an average of 14.7 overlying silver grains (Table 1) and that, in embryos enclosed within yolk sacs, 48% of the neural nuclei labeled with a mean of 7.2 grains. This 44% reduction in frequency and 51% reduction in intensity of labeled nuclei has demonstrated that the visceral yolk sac presented an incomplete barrier to passage of thymidine. This barrier effect explains at least in part the finding by Atlas et al. (1960) that tritiated thymidine given to mice before nine days of gestation did not reach the embryo. Permeability of the Visceral Hydroxyurea

Yolk Sac to

Armed with evidence that thymidine penetrated the visceral yolk sac in vitro, it was then possible to test the yolk sac for permeability to hydroxyurea. Runner (1972) showed in a preliminary report that exposure of the excised and encapsulated 8.5-day mouse embryo to hydroxyurea resulted in an BO-88%decrease in the amount

MILLER AND RUNNER

of tritiated thymidine that was incorporated into DNA. Miller (1974) and Miller and Runner (submitted) reported that, in the 8.5-day mouse embryo, hydroxyurea lowered the tissue specific rates of proliferation. The present report has shown, (Table 1) that exposure to hydroxyurea lowered the frequency with which nuclei of the neural epithelium incorporated thymidine by 15% and lowered the intensity with which nuclei were labeled by 37% irrespective of whether yolk sac was present or absent. These results indicate that the murine visceral yolk sac is readily permeable to hydroxyurea. Labeling with thymidine, on one hand, and then inhibiting labeling with hydroxyurea, on the other hand, has demonstrated the relative permeability of visceral yolk sac to thymidine and to hydroxyurea. Interposition of the visceral yolk sac decreased the effectiveness of labeled thymidine by 44 and 51% and the effectiveness of hydroxyurea by 15 and 37%. The lesser retardation of label by hydroxyurea then by yolk sac shows that the visceral yolk sac is more permeable to hydroxyurea than to thymidine. Permeability Unaffected by HydroxyureaInhibited Yolk Sac Epithelium The design of the experiment predicated that while the neural epithelium was serving as an assay for inhibition by hydroxyurea, the visceral yolk sac was itself subjected to hydroxyurea. It became necessary to inquire if inhibition of incorporation of thymidine or side effects of hydroxyurea might alter permeability of the yolk sac epithelium and thereby obscure the assay. Hydroxyurea was shown to inhibit the labeling index in endodermal epithelium of the yolk sac by 44% and the mean number of grains per nucleus by 77%. These data show that labeling of endodermal nuclei of the visceral yolk sac, as expected, was inhibited by hydroxyurea. Response of the yolk sac to hydroxyurea suggested that incorporation of thymidine

Permeability of Yolk Sac

79

in nuclei of the neural epithelium may have been inhibited by two influences that were mediated by hydroxyurea, viz., altered neural epithelium and altered yolk sac. Although the inhibitory effect of hydroxyurea on the neural epithelium in the presence of yolk sac was definitely established, the proportional inhibitions, 44.2 and 43.9% of nuclei and 51.1 and 51.7% of grains, with and without the yolk sac, respectively, were very similar. This shows that the effect of inhibited yolk sac on the incorporation of thymidine into neural epithelium had not been superimposed as a second barrier. In fact, no evidence was found that the inhibited or noninhibited yolk sac membrane offered any permeability barrier to hydroxyurea. Mother-Embryo

Transport

The visceral yolk sac is the innermost of the barriers lying between the maternal blood supply and the fluids which bathe the embryo. The finding that this membrane shows differential permeability to thymidine and to hydroxyurea sheds light on the long-standing problem of what the maternal organism actually provides for the postimplantation embryo. Nucleoside precursors are convenient and widely used for assessing incorporation into nucleic acids and for measuring rates of synthesis. It is generally accepted that nucleosides are readily taken up by cells but nucleotides are not. The consequences of competition between endogenous synthesis of nucleotides, i.e., pool size, and exogenous procurement to form nucleotides from thymidine and other nucleosides are sometimes conveniently ignored because the salvage pathway is usually thought to take preference to sustaining endogenous synthesis. Explanted embryos can sustain macromolecular synthesis for several days in the absence of exogenous nucleosides. This shows conclusively that most cells, including all embryonic tissues tested, can endogenously provide nucleic acid precursors for their rapid doubling-times. The

80

DEVELOPMENTAL BIOLOGY

question remains unanswered about the extent to which the maternal organism does in fact provide nucleoside precursors to the embryo. The finding that in the mouse embryo exogenous thymidine is transported relatively slowly as compared to a molecule like hydroxyurea suggests that at 8.5 days the embryo may have little access to exogenous thymidine as a source of precursors for nucleic acids. This view is consistent with the results following attempts to label the 8-g-day mouse embryo by injections of thymidine into the mother. Such attempts at 8.5 days have invariably failed (Atlas et al., 1960). The reason for failure to label with thymidine, at least in part, appears to be related to transport barriers provided by the yolk sac. The finding that hydroxyurea is transported more readily than thymidine from outside to inside the yolk sac membrane conforms to reports on cytotoxicity caused by the drug. Hydroxyurea given to the 12-day pregnant rat was detected in embryos and was seen to induce cell death in 3-5 hr (Scott et al., 1971; Ritter et al., 1973). Both hydroxyurea and analogs of nucleosides resulted in severe inhibition of incorporation of thymidine into acidinsoluble fractions. Since cytotoxicity resulting after treatment with hydroxyurea appeared earlier than cytotoxicity resulting from analogs of nucleosides, Scott et al. seem to have uncoupled cytotoxicity from inhibition of synthesis of DNA. The finding that the visceral yolk sac is readily permeable to hydroxyurea supports the view that, in contrast to secondary effects mediated by the maternal organism, hydroxyurea is available to and can have a primary and early effect directly on the implanted embryo. The LO-12-somite embryo, and subsequent stages of development, explanted but encapsulated within its visceral yolk sac membrane, is especially adapted for studying kinetics of substrate utilization,

VOLUME 45, 1975

for distinguishing between facultative and obligatory precursors, and for investigating the protection from and mediation of passage of noxious environmental materials. REFERENCES ATLAS, M., BOND, V. P., and CRONKITE, E. P. (1960). Deoxyribonucleic acid synthesis in the developing mouse embryo studied with tritiated thymidine. J. H&o&em. Cytochem. 8, 171-181. BECK, F., LLOYD, J. B., and GRIFFITHS, A. (1967). Lysosomal enzyme inhibition by trypan blue: A theory of teratogenesis. Science 157, 1180-1182. CLARKSON, S. G., DOERING, J. V., and RUNNER, M. N. (1969). Growth of postimplantation mouse embryos cultured in a serum-supplemented, chemically defined medium. Teratology 2, 181-186. COLE, R. J., and PAUL, J. (1966). The effects of erythropoietin on haem synthesis in mouse yolk sac and cultured foetal liver cells. J. Embryol. Enp. Morphol. 15, 245-260. KAUFFMAN, S. L. (1966). An autoradiographic study of the generation cycle in the ten-day mouse embryo neural tube. Exp. Cell Res. 42, 67-73. KAUFFMAN, S. L. (1968). Lengthening of the generation cycle during embryonic differentiation of the mouse neural tube. Exp. Cell Res. 49, 420-424. KERNIS, M. M. (1971). Abnormal yolk sac function induced by chlorambucil. Erperientia 27, 1329-1331. KOPRIWA, B. M., and LEBLOND, C. P. (1962). Improvements in the coating technique of radioautography. J. Histochem. Cytochem. 10, 269-284. MILLER, S. A. (1974). Pattern of short-term, in vitro 3H-thymidine labeling in the tissues of the 8 %-day mouse embryo. Anat. Rec. 178, 418-419. MILLER, S. A., and RUNNER, M. N. (1975). Evidence for tissue-specific proliferation rates in the lo- to 12.somite mouse embryo. Alteration by acute exposure to hydroxyurea. (submitted) RITTER, E. J., SCOTT, W. J., and WILSON, J. G. (1973). Relationship of temporal patterns of cell death and development to malformations in the rat limb. Possible mechanisms of teratogenesis with inhibitors of DNA synthesis. Teratology 7, 219-226. RUNNER, M. N. (1972). Mechanism by which the mouse embryo responds to hydroxyurea. Teratology 5, 266. SAUER, F. C. (1935). Mitosis in the neural tube. J. Comp. Neural. 62, 377-405. SCOTT, W. J., RITTER, E. J., and WILSON, J. G. (1971). DNA synthesis inhibition and cell death associated with hydroxyurea teratogenesis in rat embryos. Deuelop. Biol. 26, 306-315. SHEPARD, T. H., TANIMURA, T., and ROBKIN, M. A. (1970). Energy metabolism in early mammalian embryos. Develop. Biol. Suppl. 4, 42-58.