DESCRIPCION DE METODOS POR OBJETIVOS ESPECIFICOS.

embryos

Activation of protein kinase C (PKC) is a potential consequence o f increased flux

through the inositol/lipid cycle since diacylglycerol (DAG), a product o f the inositol/lipid cycle, is a physiological activator of PKC. In order to test whether activation of protein kinase C could influence closure of the PNP, curly tail embryos were treated in vitro

with a DAG analogue, 12-0-tetradecanoyl-phorbol-13-acetate (TPA). Unlike DAG which is rapidly metabolised, TPA, a phorbol ester, is metabolically stable and can thus cause sustained activation of PKC (Castagna e ta l, 1982; Nishizuka, 1988).

TPA treatm ent for 24 hours is lethal to embryos in vitro

Embryos were cultured from 9 days 6 hours of gestation in non-dialysed rat serum with TPA added to a concentration of 100 nM (typically used in cell culture) or 500 nM (Figure 4.1). Exposure to TPA for 24 hours was lethal to embryos at this stage (n= 6

embryos for each concentration). This is probably due to a teratogenic effect of TPA on the yolk sac which, following culture, was found to be smooth, lacking circulation and showed abnormal separation of the two cell layers. This result correlates with

observations in rat embryos (Huber and Brown, 1983), in which exposure to TPA in embryo culture causes a progressive separation of the endodermal and mesodermal cell layers.

Short-term TPA treatm ent is not teratogenic to embryos in vitro

The lethality of 24 hours exposure of embryos to TPA can probably be attributed to the fact that prolonged TPA treatment causes depletion o f PKC due to proteolysis (Young

e ta l, 1987; Nishizuka, 1988). In order to overcome this problem, TPA exposure was reduced to one hour after which the embryos were rinsed in PBS, the culture serum was changed, and culture was continued in the absence o f TPA (Figure 4.1). A one hour TPA treatment, after either 1 or 17 hours of culture, was not teratogenic to either embryo or yolk sac; there was a vigorous yolk sac circulation at the end o f the culture period and no abnormalities, other than an enlarged PNP, were observed.

TPA treatm ent mimics the effect of inositol

TPA treatment caused a significant reduction in mean PNP length at the 30-31 somite stage compared with development in whole serum (Table 4.2a, Figure 4.3). The difference in PNP length is also significant in comparison of the pooled somite stages (Table 4.2), however, although the P value is more significant for the pooled samples this is due to greater sample numbers. The percentage reduction in PNP length is greater in comparison o f embryos at the 30-31 somite stage following both inositol and TPA treatment, supporting a related mechanism of prevention o f NTD.

The protective effect of TPA treatment on closure o f the PNP appeared dose- and possibly time-dependent. The most significant reduction in PNP length was observed following treatment with 500 nM TPA after 17 hours o f culture (Table 4.2a) although the effect was not statistically different from that achieved following treatment after 1

hour. The effect o f 100 nM TPA after 1 hour of culture was reduced compared with 500 nM TPA and treatment after 17 hours of culture had no protective effect on closure of the PNP. These data suggest that the more sensitive period for TPA treatment is 9 days 7 hours of gestation (after 1 hour of culture) since the lower concentration, 100 nM, has an effect on closure of the PNP at this time point but does not at 9 days 23 hours. However, this conclusion is not absolute since raising the concentration o f TPA to 500 nM eliminates any time-dependence of the effect of TPA treatment.

Culture in the presence of 50 pg/ml inositol (Chapter 3) was repeated in parallel with the TPA cultures although in this case inositol (50 pg/ml) was added to whole serum rather than dialysed serum. Once again, there was a significant protective effect o f 50 pg/ml inositol at the 30-31 somite stage with a similar degree of protection as was seen with TPA exposure (Table 4.2a, Figure 4.3).

Table 4.2 Development of curly tail embryos (all somite num bers pooled)

cultured fo r 24 hours in the presence of 50 pg/ml inositol or with TPA treatm ent after 1 or 17 hours (mean values ± SEM)

Treatment Number o f embryos Crown-rump length (mm) Somites | PNP length (mm) R at serum 1 69 3.83 ±0.04 29.26 ±0.06 1 0.64 ±0.03 50 pg/ml inos. 67 3.88 ±0.04 29.15 ±0.17 1 0.53 iO.03 500 nM TPA (1 hr) 28 3.98 ±0.06 29.68 ±0.18 I 0.55 ±0.05 100 nM TPA (1 hr) 32 3.86 ±0.05 29.38 ±0.23 1 0.54 ±0.03 500 nM TPA (17 hrs) 1 72 3.86 ±0.04 29.32 ±0.15 1 0.48 ±0.02 100 nM TPA (17 hrs) 32 3.89 ±0.06 29.5 ±0.26 1 0.56 ±0.03 Statistically _ _{N o a N o b} Yesb* significant P= 0.4942 P= 0.6099 i P= 0.0009 (P<D.05) i F= 0.881 H =3.59 1 H= 20.9 ^ = One Way Analysis of Variance, ^ = Kruskal-Wallis One Way Analysis o f Variance on Ranks.

* = Mann-Whitney Rank Sum Test shows a significant difference between the mean PNP length of embryos cultured in whole serum and the values for embryos cultured in serum supplemented with 50 pg/ml inositol (P<0.0001, T= 3823.5) or treated with 500 nM TPA after 17 hours (P= <0.0001, T= 5929.0) or 100 nM TPA after 1 hour (P= 0.0124, T= 1289.0) or 17 hours (P= 0.0463, T= 1358.5).

Table 4.2a Development of curly tail embryos to the 30-31 somite stage following culture for 24 hours in the presence of 50 pg/ml inositol or with TPA treatm ent after 1 or 17 hours (mean values ± SEM)

Treatment Number o f \ embryos j Crown-rump length (mm) Somites PNP length (mm) R at serum 32 ! 4.00 ±0.05 30.44 ±0.09 0.61 ±0.04 50 pg/ml inos. 30 1 4.06 ±0.06 30.37 ±0.09 0.45 ±0.03 500 nM TPA (1 hr) . 1 4.05 ±0.07 30.28 ±0.11 0.46 ±0.06 100 nM TPA (1 hr) ₁₇ 3.96 ±0.09 30.35 ±0.12 0.48 ±0.03 500 nM TPA (17 hrs) 41 4.02 ±0.04 30.29 ±0.07 0.42 ±0.03 100 nM TPA (17 hrs) 19 1 4.04 ±0.06 30.53 ±0.12 0.56 ±0.04

Statistically No» Nob Yes»*

significant P= 0.9029 P= 0.5039 P= 0.0031 (P<0.05) F= 0.316 H =4.32 F= 3.76 ^ = One Way Analysis o f Variance, ^ = Kruskal-Wallis One Way Analysis of Variance on Ranks.

* = Using t-tests for pairwise comparison, the mean PNP length for culture in serum is significantly higher than the values for embryos cultured in serum supplemented with 50 fig/ml inositol (P= 0.0034, t= 3.05) or treated with 500 nM TPA after 1 hour (P=

0.0336, t= 2.19), 500 nM TPA after 17 hours (P= 0.0003, t= 3.76) or 100 nM TPA after 1 hour (P= 0.0343, t= 2.18).

Figure 4.3 Mean PNP length of embryos at the 30-31 somite stage following culture in serum supplemented with inositol or TPA. Embryos were cultured in serum alone, serum supplemented with 50 pg/ml inositol for the entire 24 hours or serum with TPA added for one hour after 1 or 17 hours (see Figure 4.1 for experimental

strategy)

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TPA treatment does not alter growth of embryos in vitro

It is important to determine whether TPA influences closure o f the PNP through non­ specific retardation of growth in culture which is known to cause a reduction in mean PNP length in curly tail embryos (Copp et a l, 1988b). This is particularly relevant in view o f the observed teratogenic effect of long-term TPA treatment.

A one hour exposure to TPA had no significant effect on parameters of growth (crown- rump length) or developmental progression (number o f somites) in curly tail embryos (all somite numbers pooled) following culture (Table 4.2). Moreover, the addition o f 50 pg/ml inositol to whole serum culture did not significantly affect crown-rump length or somite number (Table 4.2) confirming the previous finding for inositol added to dialysed serum (Table 3.1). A similar finding emerged when only embryos with 30-31 somites were considered, the stage of primary interest in this study (Table 4.2a). Therefore, the action of TPA does not appear to result from non-specific growth retardation.