a study of the effects of trypan blue injected into amphibian zygotes
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Roux' Archly f/ir Entwieklungsmeeha, nik 155, 424--428 (1964)
Department of Zoology, University of Peoria, Poona 7, India
A STUDY OF THE EFFECTS OF TRYPAN BLUE IN JECTED INT0 AMPHIB IAN ZYGOTES
M. S. LAKSHMI and GAJANAN V. SHERBET
(Received February 29, 1964) i
The teratogenic effects of t rypan blue on mammal ian embryos were studied by injecting the dye into pregnant animals (see WADDI~OTO~ and CA~T~.~, 1952, 1953; HAMBURGH, 1952, ]954). A wide spectrum of abnormal i t ies was caused. WAD])I~GTO~ and PE~u (1956) f ramed some important questions which formed the basis of their s tudy of the effects of the dye on amphib ian embryos. For example, it was not clear if t rypan blue exerted one pr imary effect or many different and unspecific ones. I t was Mso not clear whether it acted direct ly on the mammal ian embryos or its act ion was mediated through some alterat ions in sub- stances received by them from the mother. WADD1-NGTON and P~n~Y, therefore, placed early amphib ian embryos in t rypan blue solution and studied their development. In the present experiments we have injected t rypan blue solution into ferti l ized eggs of Rana and have studied its effects on their development.
Material and methods
A 1% aqueous stock solution of trypan blue (diamine blue 3B from Allied Chemicals Corporation, New York) was diluted by 1/10 Holtfreter solution (HF) to 0.1% and microinjected (0.1--0.2 #l/egg) by the procedure adopted by MA~X~RT and URsPgv~o (1963) into zygotes of Rana obtained by the method of RvG~ (1934), about 30 minutes ~fter fertilization. In the control series zygotes were injected with a solution in which 1/10 HF was mixed with distilled water instead of the stock solution of tryp~n blue. After injection the control and experimental embryos were allowed to grow in 1/10 HF at 180 C for a few hours and then changed into I-IF solution modified by BROWn and CASTON (1962). A control series of uninjected embryos was Mso maintained. Observations were made every 24 hours and the development of the embryos was recorded. The embryos were under observation until the controls developed to stage 25 (S~v~wAY, 1954). Some important cases of malformation were fixed in Bouin's fluid, cleared in cedar wood oil and sectioned for histological examination. Even during the experiments ~ccurate observations were made by anaesthetizing the embryos with MS 222 at a low concentration (o.ooo16%).
Effects of trypan blue injected into amphibian zygotes 425
Experimental results In this series 525 zygotes were injected with t rypan blue and 312
were injected with the control solution. A high rate of morta l i ty was found in both exper imental and control series (59.5 and 41% ). In both, a few embryos showed part ia l cleavage (i.e. only a part of the egg cleaved) or block of development at stage 9 to 11. These effects pre- sumably are a result of the in jury involved in the injection procedure. Exogastru lat ion occurred in three embryos of the t rypan blue series. Ten exper imental embryos developed normal ly unti l they reached the gastrnla stage; subsequently they elongated in the manner of a neurula. However, there was no sign of differentiation and on examinat ion after 24 hours they seemed to be featureless living masses. The sections of these embryos confirmed the absence of any differentiation. This might be suppression of gastrulat ion described by ~r and PEl t ry . 29.7% of the t rypan blue injected embryos and 50.3% of the control embryos developed beyond stage 14. Of these, the t rypan blue-injected ones showed head, t runk and tai l abnormalit ies, but in a rather low percentage. In the controls only one embryo was abnormal (details given in the Table). Even the malformed embryos developed normal ly unti l neurulat ion started. Degeneration of the neural tissue and dis- organized brain was observed in some eases. The notoehord was found to be normal in the embryos that were studied in sections.
Table. E//eet o/trypan blue injections on development c
Number of embryos at the beginning . . . . . . . . .
Mortality . . . . . . . . . . Partial cleavage . . . . . . . Block of development at st. 9--11 Exogastrulae . . . . . . . . . Undifferentiated . . . . . . .
Uninj ecte4 controls
189 25 1
312 128 17 10
Trupan blue injected
525 313 25 19 3
10 Embryos living beyond st. 14 . . 163 157 155
(86.2%) (50.3%) (29.7%)
Head abnormalities Microcephaly . . . . . . . . Absence of eyes (both or one) Suckers fused . . . . . . .
Trunk abnormalities Oedema and abdominal blebs Absence of gills . . . . . . . Absence of somites . . . . .
Tail abnormalities Taft absent or short . . . . . Spina bifida . . . . . . . . Gyre . . . . . . . . . . .
5.12 1.92 2.56
8.32 8.96 1.92
5.12 3.20 1.92 28*
426 M.S. LAXSHMI and GAJA:~A~r V. S]tE~BET :
Trypan blue has been shown to possess teratogenic properties by many research workers. WADDINGTO and PE~Y (1956) did not find any coloration of the amphibian embryos placed in a 0.025% solution of trypan blue presumably because only a small amount of the dye was absorbed. I t was felt that the method of injecting substances into the zygote, which one of us (M.S.L.) had the good fortune to learn in Prof. C. L. MA~Kn~T'S laboratory in the Johns Hopkins University, U.S.A., might provide some information regarding the teratogenie action of the dye.
We must concede at the outset that the high mortality produced even by control injections makes an unequivocal interpretation of the results rather difficult. Nevertheless, it seems significant that 1) the trypan blue-injected embryos showed much greater mortality than the controls; 2) exogastrulation was rare and 3) abnormalities in embryos which grew beyond stage 14 began to appear only after neurulation and incidence of abnormalities was low; but the spectrum of abnormalities except mesodermalization of notochord recorded by WADDTNGTOS and P~n~v was observed.
There is no doubt that the effects described are a result of trypan blue injections because control embryos showed none of these. The reaction shown by different vertebrate embryos to the dye seems to vary considerably. WADDYL~GTON and P~u observed the occurrence of mieroeephaly as we have noticed in these experiments. But HAm- SURG~ (1952, 1954) found a hypertrophy of the nervous system. In chick embryos no neural defects were evident after culturing them in vitro in the presence of trypan blue (MuL~RKA~, 1960), and predomi- nantly the somites were found to be affected and also the notochord. Mesodermalization of the notochord was reported by WADD~CGTO~ and Pr~Y also but we observed no mesodermalization. If the variation in the response were quantitative, we could presume that the different samples of trypan blue contained different amounts of the active frac- tion because B~CK and LLOYD (1963) have shown that the blue frac- tion of commercial samples of trypan blue was the teratogenic com- ponent and is mixed with other nonteratogenic fractions. But the results of the earlier studies with this dye in many strains of mice, rabbit, many species of amphibia and the chick do not allow one to easily sub- scribe to this view. I t is quite possible that the variation is brought about by differences in the method of application of the dye. WADDrSG- TOS and Ps~y observed a high incidence of exogastrulation. As pointed out by them this could be due to alterations in the physical state of proteins in the cytoplasm, in our opinion of the cortex. We have no compelling evidence to suggest this ; but the tow number of exogastrnlae
Effects of trypan blue injected into amphibian zygotes 427
produced in the present experiments could be attributed to this. Trypan blue was found to cause an increase in the viscosity of proteins (see WADDI~GTO~ and P~n~y, 1956). This action is said to be similar to that of lithium which has been found to cause exogastrulation or irregular gastrulation in Limnaea stagnalis (RAVEIr 1952); Planorbi8 exustus (Sm~B~T and LAXSnMI, 1963) and the sea urchin embryos (GuSTAFSO~ and WOL]~E~, 1961). In fact, GUSTArSO~ and WOL~E~T believe that exogastrulation in the sea urchin at least is due to alterations in certain properties of the cells which are responsible for the morphogenetie movements. Since trypan blue is injected into the egg, the dye pre- sumably acts and brings about changes in the state of proteins and thus arrests development long before the morphogenetic movements begin. But some embryos developed despite injections of the dye. This could be due to administration of an incomplete dose. Then a dilution of the dye would have taken place in the cortical region of the egg which led to disturbances in the morphogenetic movements and ultimate mal formation. Perhaps this is the reason why eggs which were placed directly in trypan blue solution showed irregular morphogenetic move- ments and abnormMities in a high frequency. Thus the morphogenetic disturbances themselves seem to be a result of changes in the physical state of proteins in the peripheral regions of the egg. The malformation might be a result of the irregular morphogenetie movements.
Summary Trypan blue was injected (0.001--0.002 mg/egg) into zygotes of Rana,
The trypan blue-injected series showed much greater mortality than the controls. A probable suppression of gastrulation was seen. 29.7% of the dye-injected and 50.3% of the control embryos developed beyond stage 14. Abnormalities seemed to appear only after neurulation began. Microcephaly, trunk and tail abnormalities occurred though in a small numbe