in vitro/in vivo comparison of yolk-sac function and embryo development

Toxic. in Vitro Vol. 6, No. 1, pp. 1-6, 1992 0887-2333/92 $5.00 + 0.00 Printed in Great Britain Pergamon Press pie

IN VITRO/IN VIVO COMPARISON OF YOLK-SAC FUNCTION AND EMBRYO DEVELOPMENT

J. E. ANDREWS*t, M. EBRON-McCoY*, R. M. ZUCKER:~, K. H. ELSTEIN:~ and J. M. ROGERS* *United States Environmental Protection Agency, Health Effects Research Laboratory, Developmental Toxicology Division, Research Triangle Park, NC 27711 and ~/Mantech Environmental Technology, Inc.,

Research Triangle Park, NC 27709, USA

(Received 20 December 1990; revisions received 20 March 1991)

Abstract--The yolk-sac function and development of rat embryos grown/n vitro for 24 hr, starting on day 10.5, were compared with those of embryos grown /n utero. The embryos grown in vitro had significantly fewer somites, shorter crown-rump length and smaller yolk-sac diameter when compared with the embryos grown in vivo but all values were within the normal range for this stage of gestation. Head length was not significantly different between the two groups. The cellular and nuclear volumes (Coulter counter) of nucleated yolk-sac red blood cells did not differ significantly between the two groups. RBC cell-cycle analyses by flow cytometry did not reveal any difference between/n vitro and in vivo embryos. The clinical chemistries of embryo-yolk-sac homogenates were compared. Protein, triglyceride, lactate dehydrogenase, cholesterol, urea nitrogen and glutamic--oxalacetic transaminase concentrations did not differ significantly between the two groups. The in vitro embryos had significantly lower y-glutamyl transferase (GGT) and sorbitol dehydrogenase activities. GGT activity is almost entirely in the yolk sac in the day 10.5 conceptus. ~t-Foetoprotein is synthesized by the yolk sac at this stage of development and was significantly lower in the in vitro embryos. Transferrin is transported across the yolk sac to the embryo and was significantly higher in the in vitro embryos. These data indicate that impaired yolk-sac function could, in part, be responsible for the developmental delays and the short survival times of cultured embryos.

INTRODUCTION

Whole embryo culture (WEC) has been proposed as an in vitro screen for compounds that may affect embryo development (Schmid et al., 1983) and is widely used to study the mechanisms of teratogenesis. Organogenesis-stage embryos with intact visceral yolk sacs can be conveniently cultured for 24-72 hr (New, 1978; Sanyal, 1980), and compounds that are toxic to the embryo in utero have been shown to have undesirable effects on the embryo in WEC (Schmid et al., 1983). However, the development of conceptuses in the WEC system for longer periods of time, with continued growth and differentiation, has not been possible (Sanyal, 1980). The lack of continued growth has been attributed partially to poor development of the chorioallantoic placenta in vitro and has thus been linked to improper nutritional status.

Before the formation of the chorioallantoic placenta, all embryonic nutrient uptake takes place in the extra-embryonic membranes. The principal site for the processing of embryonic nutrients is the visceral yolk sac (VYS) epithelium, which maintains histio-

1"To whom all correspondence should be addressed. Abbreviations: AFP=ct-foetoprotein; BUN=blood urea

nitrogen; EIA = enzyme immunoassay; GGT = y-glutamyl transferase; GOT = glutamic--oxalacetic transaminase; LDH = lactate dehydrogenase; PBS = phosphate buffered saline; PBST = PBS containing 0.05% Tween 20 (v/v); PI = propidium iodide; RBC = red blood cell; RIA = radioimmunoassay; SDH = sorbitol dehydrogenase; TF = transferrin; VYS = visceral yolk sac; WEC = whole embryo culture.

trophic nutritional function throughout prenatal life (Beck et al., 1967). Several investigators have examined the uptake, transport or hydrolysis of proteins (Freeman and Lloyd, 1983a; Freeman et aL, 1981; Huxham and Beck, 1985; Jollie, 1986; King and Enders, 1970; Kugler and Miki, 1985; Livesey and Williams, 1979), uptake of amino acids (Thoene et al., 1985), transport of transferrin (TF; Huxham and Beck, 1985; McArdle and Priscott, 1984; Thiriot-Hebert, 1987), transport of hexose (Koszalka et al., 1988) and synthesis of proteins (Gitlin and Perricelli, 1970; Janzen et al., 1982; Sklan and Ross, 1987) by the yolk sac. The interruption of normal histiotrophic status results in teratogenesis, as has been shown with trypan blue (Rogers et al., 1985; Williams et al., 1976), ethanol (Steventon and Williams, 1987), leupeptin (Freeman and Lloyd, 1983b) and anti-yolk-sac sera (Freeman and Brown, 1986).

Gupta et al. (1982) found that there were morpho- metric changes in the visceral endoderm of yolk sacs cultured in vitro. Miki and Kugler (1984), using histochemical methods, studied membrane-bound, cytoplasmic and mitochondrial enzymes in the VYS epithelium of in vitro and in vivo embryos on days 9.5-12.5 of gestation. Although they concluded that the enzymes involved in the digestion and energy metabolism of the two groups were comparable for up to 48 hr, they found that the membrane-bound enzyme activities were weaker in the embryos developing in vitro. Incubations for more than 48 hr resulted in significantly lower activities of many enzymes compared with development in vivo. This work indicated that yolk-sac function might be compromised during in vitro embryo culture.

1 TIV 6/I--A

2 J.E. ANDREWS et al.

Another important function of the yolk sac during the early embryo stage, in both humans (Migliaccio et al., 1986) and rats, is red blood cell (RBC) production. RBCs produced in the yolk sac are large nucleated cells whereas later gestational and adult RBCs produced in the liver are smaller and non- nucleated and therefore easily distinguishable (Zucker, 1970).

The present study was undertaken to compare yolk-sac function in embryos grown in WEC for 24hr, from day 10.5 of gestation, with that in embryos at a similar stage grown in utero. To assess comparative VYS function in vitro and in vivo, we examined: (1) concentrations of ~-foetoprotein (AFP), a protein synthesized exclusively by the VYS at this stage of development (Beck et al., 1984; Janzen et al., 1982); (2) TF, a protein transported intact across the VYS from maternal serum (Huxham and Beck, 1985); (3) the cell cycle and morphology of embryonic blood cells, which are produced in blood islands of the VYS at this stage of development (Zucker, 1970); and (4) a number of clinical chemistry parameters related both to embryonic nutrition [7-glutamyl transferase (GGT) found almost entirely in the yolk sac (Miki and Kugler, 1984) and sorbitol dehydrogenase activities] and to embryonic physiology of VYS cells [triglycerides, urea nitrogen, lactate dehydrogenase (LDH), glutamic-oxalacetic transaminase (GOT)]. This latter category contains indices normally associated with liver integrity. Because many of the typical hepatic functions are carried out by the VYS at this stage of embryogenesis, these were considered potential markers of VYS integrity and function.

MATERIALS AND METHODS

Animals. Virgin Sprague-Dawley rats obtained from Charles River Laboratories, (Raleigh, NC, USA), were mated overnight in our facilities. Pregnancy was confirmed the following morning by the presence of sperm in a vaginal smear and this was considered day 0.5 of gestation. Rats were anaesthetized with ether and embryos were explanted on day 10.5 of gestation. Embryos grown in vivo were removed on day I 1.5 of gestation.

Whole embryo culture. Early somite stage embryos were explanted on day 10.5 (15somites) using a modification of the technique described by New (1978). Embryos having intact yolk sacs with Reichert's membrane removed were cultured for 24 hr at 36°C in 25-cm 2 tissue-culture flasks on a rocker platform at 19 oscillations/min. Each flask contained 10ml of rat serum and two embryos. The culture medium was gassed initially for 30 min with 40% 02, 5% CO2, 55% N2. Subsequent gassing of 2min duration occurred at 18 hr (40% O 2, 5% CO2, 55% N2) and at 24hr (95% 02, 5% CO2). At the end of the 24-hr culture period embryos were evaluated for development and dysmorphogenesis and then homogenized in 2 ml phosphate buffered saline (PBS) for biochemical analyses.

In vivo studies. Uteri were removed from day 11.5 pregnant rats under light ether anaesthesia. The embryos with visceral yolk sacs intact were dissected from the uterus in Hanks' balanced salt solution. They

were then placed in 2 ml PBS and assayed for degree of development. The embryos with their respective yolk sacs were homogenized and aliquots were taken for biochemical analyses.

Transferrin (TF) enzyme immunoassay (EIA) procedure. TF was quantitated by using an EIA procedure modified from E1 Guindi et al. (1988). Microtitre plates (96 well; Dynatech Immulon) were coated with TF by adding 200 #1 0.05 M-carbonate buffer (pH 9.6) containing 50 ng TF (Cappe16013-1440, Durham, NC, USA) to each well. A multichannel pipettor was used to add 200 ktl PBS containing 0.05% (v/v) Tween 20 (PBST), and 2% bovine serum albumin (A-7030, Sigma Chemical Co., St Louis, MO, USA) to all wells of the coated microtitre plate to prevent non-specific binding. 100/~1 TF standard or embryonic homogen- ate in PBST and 100 #1 rabbit anti-rat TF (Cappel 0113-1442; 1:2000 in PBST) were added to the appropriate wells. The plates were incubated for 1 hr and washed three times with 300/~1 PBST at room temperature and tapped briskly on paper towels. Goat anti-rabbit IgG-alkaline phosphatase (200/~1; BioRad 172-1016, Rockville Center, NY, USA) was added to each well as the second antibody and the plates were kept at room temperature for 1 hr. The plates were washed three times with PBST and tapped dry. p-Nitrophenylphosphate (200/A) in diethanolamine buffer (BioRad 172-1063) was added to each well and the plate was developed for 15 min. The reaction was stopped by adding 50 pl 2 N-NaOH. Absorbance was read at 405 nm on a BioRad 2550 EIA Reader.

A F P radioimmunoassay (RIA) procedure. AFP concentrations were determined by Dr Stewart Sell (Houston Medical School, Houston, TX, USA) using an RIA to rat AFP (Sell et al., 1973).

Clinical chemistry. Embryos with their accompany- ing fluids and membranes were homogenized in 2 ml PBS and assayed for protein, GGT, LDH, GOT, blood urea nitrogen (BUN), cholesterol, triglyceride and sorbitol dehydrogenase (SDH) with a centrifugal analyser (Centrifichem System 400).

Cellular and nuclear volume measurements. The cellular and nuclear volumes of foetal RBCs isolated from embryos grown in utero and in vitro were measured using a Coulter Counter (Model ZBI) and Channelyzer (Model 20; Coulter Electronics, Hialeah, FL, USA). Nuclei were obtained by incubation of cells (19°C) in 0.2% Nonidet P40 (Sigma N6507) in PBS for 20 min. Before volumetric analysis, both cells and nuclei were fixed in 3% glutaraldehyde (EM Science GX015305-1, Cherry Hill, N J, USA; Zucker et al., 1979).

Flow cytometry. Flow cytometric analyses were made with an Ortho 5OH cytofluorograph (Becton- Dickinson, Westwood, MA, USA) equipped with an analytical flow cell (3OH) and a 100-mW, 488-nm-line argon-ion laser (Model 75, Lexel, Inc., Fremont, CA, USA). The flow rate was maintained at 200 particles/ sec while analysing 104 nuclei/sample. For two-colour (green/red) fluorescence analyses, compensation circuitry was used to prevent signal cross-detection. Data were collected, stored and manipulated by an Ortho 2150 computer system. Nuclei were isolated from PBS-washed RBCs (5 x 105 cells per sample) by 30min incubation (19°C) in 0.2% Nonidet P-40 supplemented with 0.5mg/ml RNase A (Sigma

Rat embryos and yolk sacs 3

R4875). The lysates were then chilled on ice and stained with 50/~g propidium iodide (PI)/ml for D N A content and 1.5/~g fluorescein isothiocyanate (FITC)/ml (Sigma F7250) for protein content (Zucker et al., 1988). Doublets were excluded from analysis by PI fluorescence signal peak v. integral discrimination. The relative percentages of nuclei in the G0/G~, S and G2/M phases of the cell cycle were estimated using Multicycle (Phoenix Flow Systems, San Diego, CA, USA), a PC-based software package using the mathematical modelling algorithm of Dean and Jett (1974).

Statistics. Embryo parameters and clinical chemistries were analysed using the general linear models procedure on the Statistical Analysis System (1985). Differences between the two groups were compared by Duncan 's multiple-range test.

RESULTS

Embryo development

Embryos that were cultured in vitro developed normal yolk-sac expansion and circulation, axial rotation, forelimb buds, neural-tube closure and had normal growth parameters when compared with historical W E C controls (data not presented). Crown- rump lengths as well as somite counts of embryos grown in vivo were significantly lower than those of embryos grown in vivo (Table 1). Yolk-sac diameter was significantly smaller in the embryos cultured in vitro but head length was not significantly different.

Clinical chemistry

A F P is synthesized exclusively by the yolk sac at this stage of embryo development. A F P concentrations were significantly reduced, in the embryos cultured in vitro, to only 7 0 0 of in vivo control values (Table 2). The AFP:p ro te in ratio was significantly reduced in the in vitro group even though there was also a slight decrease in protein concentration. Trans- ferrin concentration was 45% higher in the in vitro embryos and the transferrin:protein ratio was significantly increased in the in vitro group compared with the in vivo group. G G T activity as well as SDH concentration were significantly lower (by about 29%) in the in vitro cultured embryos compared with the in vivo embryos but the G G T and SDH to protein ratios were not significantly different.

Conceptus triglyceride, LDH, BUN, cholesterol and G O T and enzyme:protein ratios were not significantly affected by the W E C (Table 2).

Table 1. Embryonic development of rat embryos grown in vitro for 24 hr starting on day 10.5 compared with that of similar embryos

grown in vivo

Yolk-sac Crown-rump Head No. of diameter length Somite length

embryos (mm) (mm) no. (ram) Embryos grown in vivo

56 4.37±0.35 3.90+__0.30 27.7+1.09 1.91±0.16 Embryos grown in vitro

49 4.00 _+ 0.46* 3.72 _ 0.33* 26.0 _ 1.87' 1.86_+ 0.18

Values are means+SD; those marked with asterisks differ significantly (Duncan's multiple-range test) from the corresponding value for the embryos grown in vivo (*P < 0.01).

Table 2. Biochemical parameters of the conccptus for rat embryos grown in vitro for 24 hr, starting on day 10.5, compared with those

for similar embryos grown in vivo

Embryos grown Embryos grown Parameter in vivo in vitro

Number of embryos 24 20 Proteinf 256 _ 14 243 ± 20 AFP 0zg) 7.5 __. 0.5 5.3 ± 0.4** AFP (,ug)/protein (mg) 30.0 ± 2.2 23.4 + 1.7* Transferrin (pg) 275 + 48 497 __. 89* Transferrin (pg)/protein (rag) 1105 _ 196 1834 + 237* GGT (U/l) 4.8 + 0.2 3.9 + 0.2** GGT U/rag protein 0.019 ± 0.001 0.017 ± 0.001 SDH (U/I) 5.2 _ 0.2 4.1 ± 0.2** SDH U/mg protein 0.021 + 0.001 0.019 + 0.001 Triglyceride mg/100 ml 2.2 + 0.1 1.8 + 0.2 LDH (U/I) 321 + 19 280 ± 17 LDH U/mg protein 1.26 + 0.06 1.27 _+ 0.10 BUN (mg/100 ml) 0.50 + 0.04 0.62 ± 0.04 Cholesterol (mg/100 ml) 0.81 ± 0.14 0.82 ± 0.19 GOT (U/I) 8.0 + 0.7 7.3 + 0.98 GOT U/mg protein 0.028 ± 0.001 0.030 _+ 0.002

tValues are for embryo and yolk sac combined. Values are means __. SEM; those marked with asterisks differ signifi-

cantly (Duncan's multiple-range test) from the corresponding value for embryos grown in vh~o (*P < 0.05; **P < 0.01).

R B C morphometry and cell-cycle analyses

The cellular and nuclear volumes of RBCs measured by electronic cell-volume analysis did not differ significantly between the two groups (data not presented). Cell-cycle analyses by flow cytometry indicated that approximately 75% of circulating blood cells in both groups were in the S-phase of the cell cycle (Table 3).

DISCUSSION

Embryonic head length, yolk-sac circulation, axial rotation, development of forelimb buds, neural-tube closure and protein concentration were not affected by explanting and culturing embryos/n vitro for 24 hr. Our findings are consistent with those of New et al. (1976) in rats, except that we found a significantly lowered somite count after 24 hr of in vitro culture. Although the number of somites was significantly lower in the in vitro embryos than in the in vivo controls, the value of 26.0 somites is within the normal range for embryos cultured during this stage of development (Brown and Fabro, 1981; Freeman and Lloyd, 1986). The values for yolk-sac diameter and crown-rump length observed in the in vitro embryos are also within normal limits for this stage of gestation.

Many of the clinical chemistry values as well as the RBC morphometry and cell-cycle analyses did not differ between the WEC and the embryos grown in vivo, suggesting that many of the functions of the yolk

Table 3. Distribution, according to phase of cell cycle, of red blood cells from embryos grown in vitro for 24 hr, starting on day 10.5, compared

with that of similar embryos grown in vivo

Percentage of ceils in phase

G0/G~ S G~/M Embryos grown/n vivo

21.0 _+ 0.7 74.5 + 0.6 4.5 + 1.0 Embryos grown/~ vitro

21.5+0.5 75.1+1.3 3.4+1.6 Values are means + SD for four embryos.

4 J.E. ANrmEWS et al.

sacs in the two groups were comparable. The DNA 'histogram' consists of G1, S and G2/M phases of yolk-sac-derived erythroid cells. Normally, the GI and G2 periods are regulatory stages that control the initiation of DNA synthesis and mitosis. In the 11-day-old developing embryos, 75% of these cells were in the S-phase in both the in vivo and in vitro groups and the DNA 'histograms' were equivalent. The equivalence of the two populations suggests that the cells grown in vitro are not adversely affected by the growth conditions. Furthermore, if hypoxia were present, one would expect to see an S-phase or G2 block similar to that observed with cells in culture (Rice et al., 1986). The fact that 75% of the nucleated yolk-sac cells are in S-phase suggests a lack of cell- cycle regulation that normally occurs in the GI and G2 stages in tissues, adult cells and tissue-culture cell lines (O'Farrel et al., 1989). This ceil-cycle regulation is apparently not in effect in early blood-cell development. This may in part be an adaptation to the high demand for new cells in the erythroid system of a developing embryo.

AFP is synthesized in the rodent yolk sac at this stage of gestation (Beck et al., 1984; Janzen et al., 1982). The function of AFP is not known, but throughout gestation AFP is the primary serum protein and is presumed to have a vital role in embryonic development. One of these functions may be the maintenance of fluid balance in the embryo. A decrease in the production of AFP could result in significant decreases in AFP concentrations in embryonal serum, with consequent loss of colloidal osmotic pressure and movement of water from the bloodstream to the extravascular tissues. Fluid im- balances in mammalian embryos can produce blisters and oedema leading to dysmorphogenesis. Grabowski (1964 and 1977) has termed this dysmorphogenetic sequence the "edema syndrome", and such fluid imbalance is recognized as a significant mechanism of teratogenesis (Wilson, 1973). The significantly lower concentrations of AFP found in the cultured conceptus at day 11.5 of gestation suggest that yolk sacs were not synthesizing this protein at the same rate in vitro as they were in vivo and thus were functioning differently to the in vivo embryos.

Up to day 11.5 of gestation, little TF is synthesized by the visceral yolk sac (Janzen et al., 1982); it is largely transported across the yolk sac from the maternal circulation. McArdle and Priscott (1984) have suggested that TF is transported across the yolk sac by a pinocytotic mechanism as well as by receptor- mediated endocytosis. The ligand would bind to the cell-surface receptor and then be internalized in membrane endocytic vesicles. Huxham and Beck (1985) demonstrated the transfer of maternally derived TF across the visceral yolk sac of rat embryos in WEC. Our finding of significantly higher concentrations of TF in the in vitro cultured conceptuses compared with the in utero controls suggests that abnormal transport of TF may have taken place. The direct bathing of the VYS in pure rat serum in vitro may allow enhanced transport of TF compared with the in vivo situation in which Reichert's membrane and the parietal yolk sac both surround the VYS. Alternatively, interference with feedback mechanisms which control embryonic TF concentrations might account for these high

concentrations. The embryo was assayed for TF together with its exocoelomic fluid and its yolk sac so it is not clear whether one or all of these compart- ments have elevated TF concentrations. It appears from these data that transport of TF by the yolk sac in vitro is enhanced resulting in abnormal embryonic TF concentrations. The embryonic TF:protein ratio was also significantly elevated in the in vitro group, demonstrating that the increases in transferrin concentrations were even higher when considering the lower total protein concentrations in the in vitro group, suggesting that TF transport is selectively enhanced. This build-up could also result from a lack of metabolism of TF or from a compromised TF clearance mechanism.

The proteases of the VYS epithelial cells are involved in embryonic nutrition. Among the known functions of one of these proteases, GGT, are the regulation of glutathione breakdown and glutathione conjugation and the transfer of amino acids across cell membranes (Braun et al., 1987). Miki and Kugler (1984) demonstrated qualitatively in a histochemical study that GGT was present on days 10.5-11.5 in embryos grown in vivo as well as in vitro, but we have demonstrated quantitatively that the activity of this enzyme is significantly less in the in vitro cultured embryos. Miki and Kugler also found that less than 5% of GGT activity in the conceptus at this stage of gestation is in the embryo. Therefore, in the present study, a 19% decrease in GGT activity in the WEC group suggests that GGT-mediated amino acid transport is probably decreased in the in vitro cultured yolk sacs.

Sorbitol dehydrogenase is involved in the inter- conversion of sorbitol and fructose and is an important liver enzyme involved in hexose metabolism (Blakley, 1951). The significant decrease in the SDH activity of the WEC embryos compared with the embryos grown in vivo suggests that the ability to metabolize monosaccharides may be compromised in the WEC embryos. Since SDH was measured in an aliquot of the embryo plus yolk sac and we do not know which compartment contains the SDH activity, this may not indicate inhibition of yolk-sac function.

Our analyses of embryonal yolk-sac blood cells indicated no differences between embryos grown in vitro and those grown in vivo, and a number of the biochemical indices that we examined also appeared to be normal in embryos grown in vitro. Conversely, the protein synthetic capability of the VYS, as evidenced by AFP production and GGT activity, was deficient. Decreased AFP concentrations in the embryonic bloodstream could account for the clear fluid-filled blisters often seen in embryos grown in WEC. Transport of TF, an iron-binding protein critical for differentiation, was also abnormal (increased) in WEC. Taken together, our results demonstrate that yolk-sac function in WEC was not equivalent to that seen in similar-stage embryos developing in utero. We have found a number of biochemical indices that are useful for assessing VYS function, and ongoing studies will further assess these indices and compare the WEC v. in utero effects of developmental toxicants on these indices of VYS function.

Rat embryos and yolk sacs

Acknowledgement--The authors thank Judy H. Richards and D'Angelis C. Baldwin for their excellent technical assistance during this study.

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in vitro/in vivo comparison of yolk-sac function and embryo development

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