Rat Conceptuses Research Articles

Chloroquine embryotoxicity in the postimplantation rat conceptus in vitro.

The embryotoxicity of the antimalarial drug chloroquine (CQ) was evaluated in vitro using the rat whole embryo culture system. CQ was found to be embryotoxic and dysmorphogenic when added directly to the culture media containing gestational day (GD) 10 rat conceptuses. Twenty-six-hr exposure to CQ elicited dose-related decreases in embryonic crown-rump length, protein and DNA contents and increases in the incidence of morphologically abnormal embryos. At 30 microM CQ, embryonic protein content was decreased to 67% and DNA content to 58% of control while the incidence of morphological abnormalities rose to 100%. Abnormal axial rotation, micro-ophthalmia, and selective cephalic hypoplasia were the most common developmental abnormalities observed. Visceral yolk sac (VYS) vasculature and blood pigmentation were also decreased in a dose-dependent manner, as was VYS DNA content (80% of control at 30 microM). VYS protein content, however, showed an alternate pattern of response, decreasing to 87% of control at 10 microM CQ but increasing to 125% of control at 30 microM. Histologic evaluation revealed that the cytoplasm of the VYS endoderm epithelium was distended due to vacuolization produced by CQ exposure. In the embryo proper, CQ inhibited cranial neural tube development and altered the morphology of cranial neural crest cells. These observations document the in vitro embryotoxicity of CQ and suggest altered VYS histiotrophic nutrition as well as direct embryonic effects as possible mechanisms of CQ embryotoxicity.

9- cis-retinoic acid: A direct-acting dysmorphogen

Experiments in vitro with cultured rat conceptuses demonstrated that 9- cis-retinoic and acid (9- cis-RA) (300 ng/mL amniotic fluid) produced branchial arch and somite defects similar to those elicited by equal concentrations of all trans-retinoic acid (all- trans-RA), but with an increase in cephalic defects that included missing optic vesicles. After conceptuses were intraamniotically microinjected with 600 ng 9- cis-RA/mL amniotic fluid on day 10 of gestation, an unusual heart defect also observed. HPLC analyses indicated that 9- cis-RA readily underwent conversion to the less active metabolite, 13- cis-retinoic (13- cis-RA), in cultured conceptuses during the first 4 hr after treatment but only after 6 hr could elevated levels of the potent dysmorphogen all- cis-RA be detecte. In separate experiments. conversion of 13- cis-RA or of all- trans-RA to 9- cis-RA could not be detected during a 6-hr embryo culture period. Endogenous levels 9- cis-RA in whole rat embryos also were below limits of detection but small quantities of this isomer could be detected in neonatal rat eye and human embryonic brain. Our present study strongly suggests that 9- cis-RA is a direct-acting dysmorhogen with probable specific target sites of action.

Glutathione Biosynthesis in the Postimplantation Rat Conceptus in Vitro

Glutathione (GSH) biosynthesis in Day 10 rat conceptuses was characterized in whole embryo culture by evaluation of net rates of GSH replenishment in whole conceptuses, embryos. and visceral yolk sacs (VYS); uptake and distribution of [35S]cysteine and [35S]methionine from the culture medium; incorporation of 35S-labeled amino acids into GSH; and efflux of [35S]GSH into the culture medium. Diethyl maleate (DEM, 500 μM) depleted intracellular GSH pools in embryo and VYS to 30% of control values within 45 min. Restoration of GSH pools in VYS began immediately and continued at a rate of 295 pmol/ conceptus/hr until GSH concentrations exceeded initial control levels at 4 hr. GSH pools in the embryo remained depleted for over 2 hr, followed by resynthesis at initial rates of 118 pmol/ conceptus/hr. [35S]Cysteine (0.2 mM) uptake from the culture medium resulted in whole conceptus accumulations that reached 1.6 nmol/conceptus. The portion of intracellular free cysteine obtained through uptake of extraconceptal amino acid was 40-90 pmol/conceptus and represented less than 20% of the total free intracellular cysteine. [35S]Methionine (0.2 mM) accumulation surpassed that of cysteine at all time points by two- to threefold. [35S]Cysteine, added 2 hr after DEM, was incorporated into GSH at rates of 126 pmol/conceptus/hr during the first hour. By 4 hr, rates of incorporation had declined to 22 pmol/conceptusfhr. L-Buthionine-[S,R]-sulfoximine (1 mM) completely eliminated incorporation of [35S]cysteinc into GSH. Net efflux of [35S]GSH into the culture medium accounted for less than 40 pmol of total GSH when measured 5 hr after DEM addition. Although effectively transported into the conceptus and readily utilized in protein synthesis, 35S from methionine was not incorporated into GSH under any conditions tested. After chemical depletion, de novo GSH synthesis occurs exclusively in the VYS. Embryonic recovery begins only after GSH pools in the VYS are replete. Prolonged embryonic GSH depletion and slower recovery rates indicate that the embryo may be selectively susceptible to chemical insult following depletion of GSH.

Cytochrome P-450-dependent biotransformation of QA 208-199 in cultured rat conceptuses

It has been shown, using the method of rat post-implantation embryo culture, that the rat conceptus metabolizes the lipoxygenase inhibitor N-hydroxy- N-methyl-7-propoxy-2-naphthalenethan-amine (QA 208-199, QAB) in vitro. The capacity to metabolize QAB and accumulate its main metabolites depends on the developmental stage and length of exposure. In the present study, pregnant Han Wistar dams were given ip injections of either Aroclor 1254 (AC), 3-methylcholanthrene (3-MC) or phenobarbital (PB) before the dissection and culture of 9.5- and 10.5-day-old conceptuses in order to identify possible inducible conceptal cytochrome P-450 species. Conceptuses preinduced in utero were exposed to QAB in vitro for 48 hr, after which metabolites in the culture medium and conceptual tissues were determined with HPLC. Embryotoxic effects were evaluated in the same conceptuses. Preinduction in utero with AC, 3-MC or PB revealed that pretreatment with PB led, after 48 hr of culture, to a statistically significantly increased impairment of embryonic differentiation in 9.5-day-old conceptuses in vitro in comparison with vehicle-induced QAB-exposed conceptuses. Preinduction with PB led to higher levels of QAA, the main metabolite in vivo, in the culture medium. In the conceptal tissues, metabolite levels were significantly lower after preinduction with AC. In vehicle-induced 10.5-day-old embryos QAB caused less embryotoxicity in comparison with 9.5-day-old ones. In all preinduced conceptuses cultured from 10.5 to 12.5 days, yolk-sac vascularization was less affected and fewer embryonic anomalies were found in comparison with conceptuses cultured from 9.5 to 11.5 days. AC and PB pretreatment caused a decreased morphological score without changes in overall growth. Analysis of QAB and its metabolites in culture media and conceptal tissues at 12.5 days showed different levels of metabolites depending on the inducer used. To find further evidence for the involvement of cytochrome P-450 enzymes in the conceptal metabolism of QAB, conceptuses preinduced in utero (3-MC or PB) were exposed to QAB in vitro and gassed during the second half of the culture period with a mixture containing 35% carbon monoxide (CO), an inhibitor of cytochrome P-450 enzymes. CO treatment led to an inhibition of conceptal metabolism of QAB in comparison with that in conceptuses cultured under normal gassing conditions (without CO). These results strongly suggest the involvement of cytochrome P-450-dependent monooxygenases in the conceptal metabolism of QAB in vitro. 10.5-day-old conceptuses, after preexposure in utero to PB, were less susceptible to QAB embryotoxicity in vitro than were 9.5-day-old ones. In addition, the response to preinduction appeared to be age dependent.

Differences in binding characteristics of rat and human transferrin by rat visceral yolk sac placenta

Previous work has shown that human serum supplemented with rat transferrin can support the normal growth of cultured rat conceptuses, but that supplementation with human transferrin has no such effect. Such results strongly suggest an hypothesis of species-specificity. This potential specificity was therefore investigated by comparing uptake, binding, competition and vectorial transport of both rat and human transferrin using two well-established systems, those of 17.5 day rat visceral yolk sacs and anembryonic yolk sacs in culture. The results of these investigations show that human transferrin displays a lower rate of uptake and lower binding affinity for the rat transferrin receptor than does rat transferrin. Human transferrin competes poorly with rat transferrin for receptor occupancy unless present in 20-fold excess. Both molecules are taken up by receptor-mediated endocytosis and are processed in a similar manner. Anembryonic yolk sac experiments show that a greater proportion of intact rat transferrin is transported to the exocoelomic fluid than is intact human transferrin. Binding analyses show a difference in binding affinities of the two molecules for the rat transferrin receptor and also that human transferrin exhibits negative cooperativity in its binding. This evidence strongly supports an hypothesis of species-specificity in the binding of transferrin to the transferrin receptor in the rat visceral yolk sac.

Strain differences in the control incidences of morphological abnormalities in the rat whole embryo culture

Since the animal supplier to this laboratory stopped carrying the rat strain used for the previous 7 yr, a number of other strains from this supplier were tested to determine their suitability for use in the whole embryo culture system. The in vitro development from prenatal days 9.5 to 11.5 of rat conceptuses of the strain Kfm:WSA (A) was compared with that of strains Ibm:WSA (B), HanIbm:WIST (C) and Ibm:RORO (D) cultured under identical methodological conditions. The parameters for conceptal growth (yolk-sac diameter, embryonic crown-rump and head lengths) after 48 hr of culture of strains A, B and D were similar. These measurements were smaller in the conceptuses of strain C than in those of strain A, but embryonic differentiation (morphological score and somite numbers) was not delayed in comparison with strain A. The overall frequencies of conceptuses with dysmorphogenic events in strains A and B were 3.1 and 4.4%, respectively. In contrast, in strains C and D 30.0 and 22.6% of the conceptuses assessed had morphologically abnormal features. Both values were statistically significantly higher than the incidence in strain A. The types and frequencies of anomalies observed were different in the different strains tested. The results from conceptuses of strain B (derived from strain A) indicate that this strain is suitable for the culture model established in this laboratory, whereas strains C and D are not useful without methodological changes or adaptations. This study indicates that strain differences should be kept in mind when setting up the whole embryo culture system.

Glutathione Oxidation and Embryotoxicity Elicited by Diamide in the Developing Rat Conceptus in Vitro

This study was performed in the rat whole-embryo culture system to investigate the effects of glutathione oxidation by diamide, a thiol oxidant, in developing rat conceptuses during early organogenesis. The effects of diamide on reduced glutathione (GSH). glutathione disulfide (GSSG), and embryotoxicity were found to be concentration and time dependent. Diamide at concentrations of 75 and 100 μM produced abnormal axial rotation (62-89%), decreased viability (to 69% by 100 μM diamide), and reduced protein and DNA content in the embryo and visceral yolk sac (VYS) when evaluated on Day 11. High concentrations of diamide (250-500 μM) resulted in 100% mortality. GSH and GSSG levels in the conceptuses were not significantly affected during 2 hr following diamide addition at concentrations of 50 to 100 μM. At concentrations of 250 and 500 μM, rapid GSH depletion (50% of control) was seen within 5 min of exposure and was followed at 5-30 min by a significant increase in GSSG relative to control values. Diamide (500 μM) exposure for only 15 min on Gestational Day 10 was sufficient to elicit malformations (53% of exposed conceptuses with abnormal axial rotation) without significant loss of viability. After 30 min of exposure to the high concentration (500 μM), viability was decreased to 71% and defects of axial rotation increased to 87% in surviving conceptuses. This indicates that events associated with initial exposure are critical for expression of toxicity. Inhibition of glutathione disulfide reductase (GSSG reductase) activities in embryo and VYS with 1,3-bis(2-chloroethyl)-1-nitro-sourea prior to diamide addition potentiated the embryotoxicity of diamide (75 μM) and resulted in corresponding reductions in GSH/GSSG ratios as determined during the first 2 hr of exposure. Inhibition of new GSH synthesis with L-buthionine-[ S, R]sulfoximine during diamide (75 μM) exposure also exacerbated toxicity compared to diamide treatment alone. These results implicate the involvement of GSH synthesis and GSSG reductase activity in mediating the embryotoxicity of diamide.

Mechanisms of glucagon-induced increases in rates of cytochrome P450-dependent pentoxyphenoxazone O-depentylation in cultured rat conceptuses

Inclusions of glucagon (1.0 or 2.0 μM, final concentrations) in the media of cultured whole rat conceptuses resulted in concentration-dependent increases in measured rates of O-depentylation of pentoxyphenoxazone in cell-free preparations of conceptal tissues. Enzymic activities were assayed 24 h after initial exposure of the conceptuses to glucagon on day 10 of gestation. Glucagon elicited increases in tissue levels of cAMP that were parallel to those produced by 3-isobutyl-1-methylxanthine over the same time period. Tissue cAMP levels were maximal after 2 h, rapidly returned to control levels and were also equal to background levels in controls after the 24 h culture period. Dibutyryl cAMP, 3-isobutyl-1-methylxanthine, theophylline, and RO201724, a cAMP-selective phosphodiesterase inhibitor, each produced 75 to 100% increases in O-dealkylase activity. Dibutyryl cGMP and two phosphodiesterase inhibitors, enoximone (cGMP-inhibited) and zaprinast (cGMP-specific), each failed to produce statistically significant increases in O-depentylase activity. The O-depentylase was tentatively identified as a conceptus-specific P450 cytochrome that is synthesized predominantly in tissues of the visceral yolk sac. The results indicated that glucagon may upregulate a unique, xenobiotic-biotransforming P450(s) via a long-term mechanism(s) specifically involving tissue cAMP.

Phosphoinositide metabolism in the developing conceptus. Effects of hyperglycemia and scyllo-inositol in rat embryo culture.

Culture of the postimplantation rat conceptus in hyperglycemic medium causes developmental abnormalities and is associated with a diminished water-soluble myo-inositol content. We investigated the effect myo-inositol depletion has on lipid-soluble phosphoinositides, precursors, and water-soluble inositol phosphates. Rat conceptuses were cultured from gestational day 9.5 (presomite, early head fold) to day 10.5 (7-15 somites) in 6.7-73.3 mM D-glucose. Significant decreases in the phosphoinositides of the embryo were observed with increased culture D-glucose concentrations. PI was reduced 15-34%, PIP 18-46%, and PIP2 26-46%. Yolk sac phosphoinositides also were reduced but to a lesser degree. Culture in hyperglycemic media also mediated significant reductions of conceptus inositol phosphates. To investigate whether effects similar to those induced by D-glucose could be mediated by another agent capable of decreasing myo-inositol content, we used scyllo-inositol, a transported but nonmetabolized isomer of myo-inositol. Conceptuses cultured in medium containing scyllo-inositol (0.06-16.7 mM) had dose-dependent decreases of myo-[3H]inositol in water-soluble and lipid-soluble fractions. Incorporation of myo-[3H]inositol into phosphoinositides and inositol phosphates was decreased concomitantly. Developmental effects of D-glucose and scyllo-inositol were assessed in rat conceptuses cultured from day 9.5 (presomite, early head fold) to day 11.5 (22-28 somites). Culture in 40.0-73.3 mM glucose and 0.06-33.3 mM scyllo-inositol impaired growth while increasing dysmorphogenesis in a dose-dependent manner. The results suggest that decreases in conceptus myo-inositol and associated diminution of phosphoinositides, which are the inositol/lipid cycle precursors, are dysmorphogenic and may contribute to the etiology of diabetic embryopathy.

Conceptal biotransformation of 4-oxo-all- tranas-retinoic acid, 4-oxo-13- cis-retinoic acid and all- tranas-retinoyl-β-glucuronide in rat whole embryo culture

In cultured rat conceptuses, intraamniotic microinjections of 2500 ng/mL of 4-oxo-13- cis-retinoic acid, 600 ng/mL 4-oxo-all- trans-retinoic acid or 4000 ng/mL all- trans-retinoyl-β-glucuronide, produce qualitatively and quantitatively similar patterns of dysmorphogenesis as those reported after the intraamniotic microinjection of 250 ng/mL all- trans-retinoic acid [Lee et al., Teratology 44: 313–323, 1991; Creech Kraft et al., Teratology 45: 259–270, 1992]. In the present study, we utilized HPLC techniques to analyze retinoid levels in cultured rat conceptuses, 1.5 hr after intraamniotic microinjections of 4-oxo-13- cis-retinoic acid (2500 ng/mL), 4-oxo-all- trans-retinoic acid (600 ng/mL) or all- trans-retinoyl-β-glucuronide (4000 ng/mL). Our findings show that, after the microinjections of4-oxo-all- trans-retinoic acid or 4-oxo-13- cis-retinoic acid (at these selected concentrations), 4-oxo-all- trans-retinoic acid was predominant in the embryos proper at concentrations of about 200 nM. This was roughly equivalent to the levels of all- trans-retinoic acid assayed after microinjections of all- trans-retinoyl-β-glucuronide (4000 ng/mL). We conclude from these studies that both 4-oxo-all- trans-retinoic acid and all- trans-retinoic acid behave as ultimate or proximate dysmorphogens.

Microinjections of cultured rat conceptuses: studies with 4-oxo-all-trans-retinoic acid, 4-oxo-13-cis-retinoic acid and all-trans-retinoyl-beta-glucuronide.

4-Oxo-all-trans-retinoic acid, 4-oxo-13-cis-retinoic acid and all-trans-retinoyl-beta-glucuronide were intraamniotically microinjected in rat embryos on day 10 of gestation and cultured until day 11.5. A comparison of the concentration-effect relationships showed that the dysmorphogenic effects produced by these metabolites were qualitatively similar to those of parent all-trans-retinoic acid. Compared with all-trans-retinoic acid (300 ng/ml), the dysmorphogenic effects were elicited by a 2-fold higher concentration of 4-oxo-all-trans-retinoic acid, an approximately 10-fold higher concentration of 4-oxo-13-cis-retinoic acid and a 16-fold higher concentration of all-trans-retinoyl-beta-glucuronide. A surplus of uridine 5'-diphospho-glucuronic acid, microinjected together with 300 ng/ml all-trans-retinoic acid, decreased the observed embryo-toxicity of all-trans-retinoic acid, suggesting the possibility of glucuronidation in tissues of the conceptus per se. The results of the study provide further support for the hypothesis that 4-oxo-all-trans-retinoic acid and all-trans-retinoic acid are, in contrast to the corresponding cis-isomers and glucuronides, ultimate dysmorphogenic retinoids.

Transfer of plutonium to rat embryos in vivo and in vitro.

The 239Pu distribution in the 12.5-day-old rat conceptus was compared between in vivo and in vitro experimental systems to establish a possible mechanism of cross-placental transfer of this radionuclide. In the in vivo study, plutonium citrate solution was injected intravenously to pregnant Wistar rats. In the in vitro study, either plutonium citrate or plutonium hydroxide colloid was administered, as a solution of Eagle MEM and FCS containing 239Pu at the concentration used in the maternal serum in the in vivo experiments, to rat conceptuses maintained by the whole-embryo culture method. The concentration of 239Pu in the yolk sac (239Pu activity per gram wet weight) were much higher than in the embryo in both the in vivo and in vitro experiments, suggesting that the yolk sac may be an effective barrier against the transfer of plutonium to the embryos. The ratios of the 239Pu concentration in the yolk sac to that in the embryo were relatively constant with time after administration in the in vitro system; 18-27 for plutonium citrate and 67-84 for plutonium hydroxide. In the in vivo experiment, these ratios changed with time after injection; 15 at 5 min and 62 and 60 min after injection. This suggests that in the in vivo system, the chemical form of 239Pu changed with time after injection, probably to a macromolecular form such as the hydroxide colloid or plutonium-protein complex although 239Pu was injected to the maternal blood as citrate.

Comparison of the metabolic activity of yolk sac tissue in the whole embryo and isolated yolk sac culture

In rat visceral yolk sac tissue cultured from 10.5 to 12.5 days of prenatal age, metabolism of the lipoxygenase inhibitor N-hydroxy- N-methyl-7-propoxy-2-naphtalinethanamine (QA 208-199, QAB) and the accumulation of its metabolites have been shown previously (1,2). In this study, the metabolic activities of visceral yolk sac tissues cultured either alone or together with the embryo were compared. The metabolite patterns in medium and visceral yolk sac tissue generated by intact conceptuses or by isolated visceral yolk sac tissues were similar. After 24 as well as 48 h of culture, the major in vivo metabolite, 7-propoxy-napthalene-2-ylacetic acid (QAA) and other, as yet unidentified metabolites, accumulated in embryo proper and visceral yolk sac tissues. QAB was not found in the embryo proper, and was only found in yolk sac tissues using the higher concentration. In particular the metabolites M5 and M6 exhibited a massive accumulation in the visceral yolk sac, whereas QAA, M3, and M4 accumulated to a much lesser degree. In isolated yolk sacs cultured for 48 h, tissue levels of QAA and M4 were similar to those in yolk sacs of cultured whole conceptuses, whereas M5 and M6 exhibited twofold higher levels in isolated yolk sacs. These findings were in agreement with the distinct increase of myeloid figures containing partly fragmented inclusion bodies in yolk sac tissues. These results suggest that the visceral yolk sac may be the major site of QAB metabolism in cultured rat conceptuses in vitro.

Distribution of 3 H in Rat Conceptus Cultured in Vitro Following Brief Administration of [ 3 H]Thymidine

Rat embryos with intact visceral yolk sacs, explanted at 12 1/2 days of gestation, were cultured in vitro for up to 60 min in medium consisting of fetal calf serum, Eagle's MEM, and [3H]thymidine (1.2 kBq ml-1), using the roller bottle method. The total amount of 3H incorporated into the conceptus during the 60-min incubation was 79.2 Bq, and approximately 33, 23, and 44% of this activity was distributed to the embryo, the yolk sac, and the fluid in the exocoelom and amniotic cavity, respectively. The rate of 3H accumulation in conceptuses decreased with time in culture. It appeared that the decrease in the viability of the conceptus was not responsible for this phenomenon. The concentration of 3H in the yolk sac, i.e., 3H activity per gram wet weight, was 2.1 times that in the medium at the end of culture. In contrast, the 3H concentration in the embryo was significantly lower than that in the medium. These findings suggest that the visceral yolk sac of rat conceptuses may act as a barrier to the transport of tritiated thymidine between the medium and embryo.

Ultrastructure and function of the rat yolk sac: Damage caused by teratogenic anti‐VYS serum and recovery

It was hypothesized that heterologous anti-rat visceral yolk sac serum (AVYS) exerts its teratogenic effect by reducing the endocytosis of serum proteins by the visceral yolk sac (VYS), thus reducing the supply of amino acids to the embryo and VYS. To evaluate this hypothesis, we studied the effect of teratogenic AVYS on the endocytic function of the VYS and the ultrastructure of the VYS and parietal yolk sac (PYS). Rat conceptuses were exposed to a teratogenic dose of AVYS on the 10th day of gestation in vivo or in vitro. Control and AVYS-exposed specimens were collected 24-192 hr later and prepared for scanning and transmission electron microscopy (SEM and TEM, respectively) utilizing standard procedures. The Endocytic Index was calculated for the VYS utilizing standard procedures. Approximately 97% of the in vivo exposed and 94% of the in vitro exposed embryos were morphologically abnormal. Ultrastructural observations showed that exposure to AVYS in vivo or in vitro caused severe damage to the VYS endodermal epithelial cells with loss of cellular borders, reduction in the number and length of microvilli, and increased cellular inclusions; and some damage to PYS endodermal cells with increased blebbling and decreased cell number. Recovery was evident at 72 hr and complete by 96 hr. The Endocytic Index was significantly reduced in the VYS 24 and 48 hr after injecting AVYS into the pregnant rat but was not significantly different at 96 and 192 hr. Our results show that the AVYS antiserum damaged visceral endodermal epithelium experienced ultrastructural recovery with parallel functional recovery. These studies suggest that transient yolk sac placental ultrastructural damage and dysfunction was probably sufficient to cause irreversible damage to the developing embryo during early organogenesis. We conclude that the proximate effect of the AVYS was on the plasma membrane of the visceral endoderm and that decreased pinocytosis is a consequence of this effect.

Sources of amino acids for protein synthesis during early organogenesis in the rat. 2. Exchange with amino acid and protein pools in embryo and yolk sac

Tenth-day rat conceptuses were cultured in whole rat serum containing [3H]leucine and harvested after 24 or 48 h. Hydrolysates of the acid-precipitable fraction of embryo or yolk-sac homogenates were prepared and subjected to paper chromatography. Liquid scintillation counting of the separated amino acids showed that leucine was the only amino acid with above-background radioactivity. This established that radiolabel was not transferred from leucine to other amino acids in the cultured rat conceptus. Tenth-day rat conceptuses were cultured in whole rat serum containing [3H]leucine, as above. After 19 h, some conceptuses were harvested; other conceptuses were rinsed, transferred to culture medium without [3H]leucine, and after a further 24 h of culture the embryos and yolk sacs were harvested. A comparison of the protein-associated radioactivity of embryo and yolk sac before and after culturing for the further 24-h period showed that these structures quantitatively conserve radiolabelled leucine incorporated into their proteins. Further experiments involved culturing the rat conceptus for 24 h as above but in the presence of either [3H]leucine or [3H]leucine-labelled serum proteins. After harvesting the conceptuses, the specific radioactivity of [3H]leucine was determined in the acid-soluble and acid-precipitable fractions prepared from embryo and yolk-sac homogenates. The specific radioactivity of [3H]leucine in the acid-soluble fraction of embryos or yolk sacs from conceptuses grown in the presence of radiolabelled protein was about 120 per cent of that in the culture medium, while that in the acid-precipitable fractions was about 70 per cent of that in the culture medium. By comparison, the specific radioactivity of [3H]leucine in the acid-soluble fraction of embryos and yolk sacs from conceptuses grown in the presence of free [3H]leucine was only 3-4 per cent of that in the culture medium, while that in the acid-precipitable fraction was about 1 per cent of that in the culture medium. If our data on the fate of leucine incorporated into newly synthesized proteins of the early post-implantation rat embryo can be extrapolated to the other amino acids, they suggest that once amino acids have been incorporated into newly synthesized protein in embryo or yolk sac, they are not exchanged to any detectable extent with amino acid pools outside the conceptus. The results also provide independent confirmation of our earlier conclusion that the only significant source of amino acids utilized by the 10th-day embryo is protein taken up by the yolk sac and digested intracellularly.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of QA 208-199 and its metabolite 209-668 on embryonic development in vitro after microinjection into the exocoelomic space or into the amniotic cavity of cultured rat conceptuses

The objective of this study was to determine the direct embryotoxic effects in vitro of N-hydroxy-N-methyl-7-propoxy-2-naphthalene-ethanamine (QA 208-199, QAB) and of one of its metabolites, 7-propoxy-naphthalene-2-ylacetic acid (209-668, QAA), after circumventing the bioconverting conceptual membranes. The compounds were, therefore, microinjected either into the exocoelomic space or into the amniotic cavity of rat conceptuses of 10 d at prenatal age at doses of up to 84.9 ng (QAA) and 180 ng (QAB) per conceptus respectively. The conceptuses were subsequently cultured for 28 h after which their development was assessed. QAB produced marginal effects on embryonic differentiation only after microinjection of the compound into the amniotic cavity. Dysmorphogenic effects, however, occurred in a dose-dependent fashion after either exocoelomic or intraamniotic microinjections of the compound. The frequencies and types of anomalies were similar after either exposure route and consisted predominantly of anomalies associated with axial rotation. QAA also impaired embryonic differentiation at only the high dose level of 84.9 ng per embryo and after intraamniotic injections only. Dysmorphogenic effects were observed in all experimental groups, although the differences were not statistically significant when compared with the concomitant controls. An increased proportion of anomalies observed were in the cephalic region as compared to the defects produced by QAB. These data suggest that QAA most probably is not the QAB metabolite responsible for the embryotoxic action of QAB in vitro. Furthermore, the results tend to confirm the suggested involvement of the visceral yolk sac membrane in mediating QAB embryotoxicity.

Quantitation and immunohistochemical localization of DNA adducts in rat embryos and associated yolk sac membranes exposed in vitro to N‐acetoxy‐2‐acetylaminofluorene (N‐Ac‐AAF)

Specific antibodies and radioimmunoassay (RIA) were used to measure the levels of acetylated and deacetylated C-8 substituted deoxyguanosine adducts in day 11 rat embryos and their associated yolk sacs after exposure of whole rat conceptuses in vitro to the teratogen N-acetoxy-2-acetylaminofluorene (N-Ac-AAF). The deacetylated adduct predominates in both the embryo and the associated yolk sac, and a dose response for adduct formation was observed when adducts were quantitated by RIA. Immunohistochemical localization of the deacetylated adducts revealed that adducts were confined to the nuclei in all tissues examined and that the abundance of adducts varied within and between tissues. Our initial findings indicate that specific DNA adduct antibodies may be useful in the study of teratogenesis induced by a wide variety of agents that modify DNA.

Cytochrome P450-dependent bioactivation of prodysmorphogens in cultured conceptuses

These investigations were undertaken to determine the extent to which tissues of cultured rat conceptuses contain cytochrome P450 isoforms in sufficient quantities to significantly influence the capacity of certain chemicals to elicit dysmorphogenic effects in vitro. Investigations with highly sensitive probe substrates/ inhibitors and with immunologic methods enabled the detection of at least four separate P450 isoforms in tissues of the visceral yolk sac, ectoplacental cone, and embryo proper. One of the isoforms was identified as P450IA1 and was found to be inducible by polycyclic aromatic hydrocarbons in all three tissues. Other isoforms exhibited properties differing from characterized adult rat hepatic isoforms. Each of the isoforms was detectable in conceptuses on gestational days 10, 11, 12, and 14 and was present in the highest concentrations in the visceral yolk sac. Conceptual P450IA1 catalyzed the conversion of dysmorphogenically inactive 2-acetylaminofluorene to 7-hydroxy-2-acetylaminofluorene, a proximate dysmorphogen. Investigations with microinjections suggested that visceral yolk sac hydroxylation was largely responsible for the bioactivation reaction in vitro. The same isoform exhibited no capacity to influence the dysmorphogenic activity of cyclophosphamide. The results demonstrated that tissues of cultured rat conceptuses may contain P450 isoforms in sufficient amounts to markedly influence the dysmorphogenic activity of substrates of the corresponding isoforms.

CAMP-dependent regulation of P450-catalyzed dealkylation in rat conceptal tissues

Exposures of cultured whole rat conceptuses to varying concentrations of dibutyryl cyclic AMP or isobutylmethylxanthine, alone or in combination, resulted in significant increases in rates of cytochrome P450-dependent depentylation of pentoxyphenoxazone in cell-free preparations. Lesser increases in rates of debenzylation of benzyloxyphenoxazone were also observed. In cultured whole conceptuses, basal depentylase and debenzylase activities in the visceral yolk sac were approximately sixfold higher than in the embryo. The ectoplacental cone and decidual tissues exhibited no detectable depentylase activity. Only the visceral yolk sac exhibited increased depentylase activity in response to dibutyryl cyclic AMP and isobutylmethylxanthine. Inhibitory antibodies raised against adult hepatic P450s IIB1, IIC11, and IA1 failed to significantly inhibit the yolk sac depentylase activities of noncultured conceptuses. The results suggested that the conceptal depentylation reaction may be catalyzed by a unique P450 isoform(s) that is not expressed during adult life.