Roux' Archly f/ir Entwieklungsmeeha, nik 155, 424--428 (1964)

Department of Zoology, University of Peoria, Poona 7, India

A S T U D Y O F T H E E F F E C T S OF T R Y P A N B L U E I N J E C T E D I N T 0 A M P H I B I A N Z Y G O T E S

By

M. S. LAKSHMI and GAJANAN V. SHERBET

(Received February 29, 1964) i

Introduction

The te ra togen ic effects of t r y p a n blue on m a m m a l i a n embryos were s tud ied b y in jec t ing the dye into p regnan t an imals (see WADDI~OTO~ and CA~T~.~, 1952, 1953; HAMBURGH, 1952, ]954). A wide spec t rum of abnormal i t i e s was caused. WAD])I~GTO~ and P E ~ u (1956) f r amed some i m p o r t a n t quest ions which fo rmed the basis of the i r s t u d y of the effects of the dye on a m p h i b i a n embryos . F o r example , i t was not clear if t r y p a n blue exe r t ed one p r i m a r y effect or m a n y dif ferent and unspecif ic ones. I t was Mso no t clear whe ther i t ac ted d i rec t ly on the m a m m a l i a n embryos or i ts ac t ion was m e d i a t e d th rough some a l te ra t ions in sub- s tances received b y t h e m from the mother . WADD1-NGTON and P~n~Y, therefore, p laced ear ly a m p h i b i a n embryos in t r y p a n b lue solut ion and s tud ied the i r deve lopment . I n t he p resen t exper iments we have in jec ted t r y p a n blue solut ion into fer t i l ized eggs of Rana and have s tud ied its effects on the i r deve lopment .

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 I n th is series 525 zygotes were in jec ted wi th t r y p a n blue and 312

were in jec ted wi th the control solution. A high r a t e of m o r t a l i t y was found in bo th exper imen ta l and control series (59.5 and 41% ). I n both , a few embryos showed pa r t i a l c leavage (i.e. only a p a r t of the egg cleaved) or b lock of deve lopmen t a t s tage 9 to 11. These effects pre- sumab ly are a resul t of the in ju ry involved in the in jec t ion procedure. Exogas t ru l a t i on occurred in three embryos of the t r y p a n blue series. Ten exper imen ta l embryos developed no rma l ly unt i l t h e y reached the gas t rn la s tage; subsequent ly t hey e longated in the manner of a neurula . However , there was no sign of d i f ferent ia t ion and on examina t ion af ter 24 hours t h e y seemed to be featureless l iving masses. The sections of these embryos conf i rmed the absence of any different iat ion. This migh t be suppression of gas t ru la t ion descr ibed b y ~r and PE l t ry . 29.7% of the t r y p a n blue in jec ted embryos and 50.3% of the control embryos deve loped beyond s tage 14. Of these, the t r y p a n b lue- in jec ted ones showed head, t r u n k and ta i l abnormal i t ies , b u t in a r a the r low percentage. I n the controls only one embryo was abnorma l (details given in the Table). E v e n the mal fo rmed embryos deve loped no rma l ly unt i l neuru la t ion s ta r ted . Degenera t ion of the neura l t issue and dis- organized b ra in was observed in some eases. The no toehord was found to be no rma l in the embryos t h a t were s tud ied 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

Injected controls

312 128 17 10

Rana

Trupan blue injected

525 313 25 19 3

10 Embryos living beyond st. 14 . . 163 157 155

(86.2%) (50.3%) (29.7%)

(per cent)

(0.63)

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 :

Discussion

Trypan blue has been shown to possess teratogenic properties by many research workers. WADDINGTO• and P E ~ Y (1956) did not find any coloration of the amphibian embryos placed in a 0.025% solution of t rypan blue presumably because only a small amount of the dye was absorbed. I t was felt tha t 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 tha t the high mortal i ty produced even by control injections makes an unequivocal interpretation of the results rather difficult. Nevertheless, it seems significant tha t 1) the t rypan blue-injected embryos showed much greater mortal i ty 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 tha t the effects described are a result of t rypan 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 hyper t rophy of the nervous system. In chick embryos no neural defects were evident after culturing them in vitro in the presence of t rypan blue (MuL~RKA~, 1960), and predomi- nant ly the somites were found to be affected and also the notochord. Mesodermalization of the notochord was reported by WADD~CGTO~ and P r ~ Y also but we observed no mesodermalization. I f the variation in the response were quantitative, we could presume tha t the different samples of t rypan blue contained different amounts of the active frac- tion because B~CK and LLOYD (1963) have shown tha t the blue frac- tion of commercial samples of t rypan 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 tha t the variation is brought about by differences in the method of application of the dye. WADDrSG- TOS and P s ~ 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 at tr ibuted 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 tha t 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 tha t 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 t rypan 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 t rypan 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 t rypan blue-injected series showed much greater mortal i ty 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, t runk and tail abnormalities occurred though in a small number. Sections of a few embryos reveMed disorganized brain and degeneration of the neural tissue. There were no eases of mesodermaliza- tion of the notochord.

The results are discussed from the view point that t rypan blue alters the physical state of proteins in the cytoplasm of the egg.

Acknowledgement. One of us (G.V.S.) gratefully acknowledges the award of a National Research Fellowship by the Government of India.

References BECK, F., and J. B. LLOYD: The preparation and teratogenic properties of pure

trypan blue and its common contaminants. J. Embryol. exp. Morph. 11, 175--184 (1963).

428 M.S. LAKSHlV~ and GAJANAN V. SHERBET: :Effects o~ trypan blue etc.

B~ow~, D. D., and J. CASTOr: Biochemistry of amphibian development. I. Ribo- somes and protein synthesis in early development of Rana pipiens. Develop. Biol. 5, 412-434 (1962).

GUSTAFSON, T.~ and L. WOLPERT: Studies on the cellular basis of morphogencsis in the sea urchin embryo. Gastrulation in vcgetalized larvae. Exp. Cell Res. 22, 4 3 7 ~ 4 9 (1961).

HA~BURG~, H. : Malformation in mouse embryos induced by trypan blue. Nature (Lond.) 169, 27 (1952).

- - The embryology of trypan blue induced abnormalities in mice. Anat. Rec. 119, 409--422 (1954).

MARKERT, C. L., Land H. U~SP~VNG: Production of replicable persistent changes in zygote chromosomes of Rana pipiens by injected proteins from adult liver nuclei. Develop. Biol. 7, 560--577 (1963).

MUL~ER]~, L. : The effects of tryp~n blue on chick embryos cultured in vitro. J. Embryol. exp. Morph. 8, 1--5 (1960).

RAVE~, Cm~. P. : Morphogenesis in Limnaea stagnalis and its disturbances by lithium. J. exp. Zool. 121, 1--77 (1952).

RuG~, 1~.: Induced ovulation and artificial fertilization in the frog. Biol. Bull. 66, 22--29 (1934).

S~E~BET, G. V., and M. S. LAKSm~I: Lithium susceptibility in Planorbis exustus. Naturwissenschaften ~0, 453 (1963).

S~u~wAY, W. : Introduction to vertebrate embryology. New York: John Wiley & Sons 1954.

WADDI~GTO~, C. H., and T. C. C~TER: Malformation in mouse embryos induced by trypan blue. Nature (Lond.) 169, 28 (1952).

- - - - A note on abnormalities induced in mouse embryos by trypan blue. J. Embryol. exp. Morph. 1, 167--180 (1953).

--, and M. M. PER~u Teratogenic effects of trypan blue on amphibian embryos. J. Embryol. exp. Morph. 4, 110--119 (1956).

Dr. M. S. LAI(s~a~, Zoologisches Institut der Universit~t, 53 Bonn a. Rh., Poppelsdorfer SchloI~