/. Embryol. exp. Morp/u, Vol. 17, 2, pp. 263-266, April 1967
With 2 plates
Printed in Great Britain
263
Reversible effect of
chloroacetophenone by sulphydryl groups on
morphogenesis of chick embryos
By LEELA MULHERKAR, 1 S. S. JOSHI, 1 B. A. DIWAN1
& P. N. JOSHP
From the Department of Zoology, University of Poona
INTRODUCTION
An effect of sulphydryl-containing substances such as cysteine and glutathione in the reduced state on the inducing capacity of post-nodal pieces has
been shown by Waheed & Mulherkar (1966). Cysteine was also shown to reverse
the effects of chloroacetophenone (CAP), a specific SH inhibitor, on the morphogenesis of chick embryos (Mulherkar, Rao & Joshi, 1965). In addition to SH
groups both cysteine and glutathione contain amino groups as well, and it is
therefore interesting to test whether the effects shown by these substances are
due to their SH or their amino groups.
Thiomalic acid contains SH groups but no amino groups, while serine contains amino groups but no SH groups. Moreover the structural formula of
serine is exactly similar to that of cysteine except that SH is replaced in serine by
OH. CAP-treated embryos were subsequently treated with each of the above
substances to see whether they bring about a reversal of the effects produced by
CAP.
MATERIALS AND METHODS
Fresh fertilized hen eggs were incubated at 37-5 °C until the definitive primitive
streak stage was reached. The glassware used in culturing the embryos was
sterilized, and the solutions employed for Pannet Compton's saline (PC saline)
were separately autoclaved. Embryos explanted by the method of New (1955) at
the definitive streak stage were treated with 5 x 10~4 M chloroacetophenone for
15min as described by Lakshmi (1962a). CAP was completely removed by
carefully giving three washes in normal PC saline. In experiment I CAP-treated
embryos were divided into two groups, experimentals and controls. The
experimental group of embryos was subsequently treated with a few drops of
1
2
Authors' address: Department of Zoology, University of Poona, Poona, 7, India.
Author's address: Department of Biochemistry, University of Poona, Poona, 7, India.
iy
j EEM
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L. MULHERKAR ET AL.
equimolar concentration (5 x 10~4 M) of thiomalic acid. The group subsequently
treated with normal PC saline served as controls.
In Exp. II the experimental group of CAP-treated embryos was subsequently
treated with a few drops of approximately equimolar concentration (4 x 10~* M)
of serine; while the control group was subsequently treated with normal PC
saline. Embryos were in all cases incubated further for about 18 h after which
they were observed and prepared for microscopical studies. About fifteen master
controls without any chemical treatment were kept in each experiment. They
were found to develop normally (Plate 2, fig. K).
In Exp. I, 33 experimentals (CAP-treated embryos subsequently treated with
thiomalic acid) and 35 controls (CAP treated embryos subsequently treated
with normal PC saline) were kept.
In Exp. II, 24 experimentals (CAP-treated embryos subsequently treated with
serine) and 26 controls treated with saline were kept.
RESULTS AND DISCUSSION
All the CAP-treated embryos in both Exps. I and II showed malformations,
mainly of the nervous system, as described previously (Lakshmi, 1962 a;
Mulherkar et al. 1965) and shown in the figures (Plate 1, figs. A, E). From
sections (Plate 1, figs. B, F) it could be seen that the nervous system is poorly
developed and the neural tube remains open at all levels. In Exp. I CAPtreated embryos subsequently treated with thiomalic acid show complete
reversal to normal development in 31 out of 33 cases (94 %) (Plate 1, fig. C).
Well-differentiated closed neural tubes can be seen in sections of these embryos
(Plate 1, fig. D).
In Exp. II, as in Exp. I, CAP-treated embryos show malformations mainly of
the nervous system (Plate 1, figs. E, F). CAP-treated embryos subsequently
treated with serine do not show reversal to normal development in any of the
twenty-six embryos tested (Plate 2, figs. G, H). The results of these experiments
can be explained on the basis of the presence or absence of SH groups in the
PLATE 1
Fig. A. Embryo treated with chloroacetophenone (CAP) at definitive primitive streak, set I.
x36.
Fig. B. Transverse section through an embryo treated with CAP at definitive primitive streak.
Fig. C. Embryo treated with CAP at definitive primitive streak followed by treatment with
thiomalic acid, x 36.
Fig. D. Transverse section of embryo treated with CAP at definitive primitive streak followed
by treatment with thiomalic acid.
Fig. E. Embryo treated with CAP at definitive primitive streak, set II. x 36.
Fig. F. Transverse section through an embryo treated with CAP at definitive primitive streak.
PLATE 1
J. Embryol. exp. Morph., Vol. 17, Part 2
*r.
E.
L. MULHERKAR, S. S. JOSHI, B. A. DIWAN & P. N. JOSHI
facing p. 264
/. Embryol. exp. Morph., Vol. 17, Part 2
PLATE 2
K
L. MULHERKAR, S. S. JOSHI, B. A. DIWAN & P. N. JOSHl
facing p. 265
Sulphydryl groups and morphogenesis
265
chemicals. Cysteine was previously reported to bring about the reversal of GAP
effect (Mulherkar et al. 1965). About fifteen CAP-treated embryos subsequently
treated with glutathione in the reduced state at a concentration of 1 x 10~5 M were
also found to show reversal in 95 % of cases (Plate 2, fig. J) as opposed to those
treated with CAP alone (Plate 2, fig. I). Both thiomalic acid and glutathione,
like the cysteine used previously, contain SH groups in their structure and therefore it is likely that the reversal of the CAP effect shown by them may be
attributed to their SH groups. On the contrary, the amino acid serine which is
similar to cysteine except that the SH of cysteine is replaced by OH, is unable
to bring about the reversal of the CAP effect.
The importance of SH groups in morphogenesis and induction has been
emphasized by Brachet (1950) and Rapkine & Brachet (1951). SH groups are
known to be important in the closure of the neural tube. Their role in the induction of the nervous system has also been shown in the post-nodal pieces of
chick embryos (Waheed & Mulherkar, 1966; Muzumdar, 1966).
The possible mode of action of SH groups in bringing about the induction of
the nervous system and morphogenesis has been discussed at length previously
(Mulherkar et al. 1965; Lakshmi, 1962a, b; Lakshmi & Mulherkar, 1963). The
present data also suggest that CAP acts on the SH groups of protein molecules
and blocks the induction and morphogenesis of the nervous system. When SHcontaining substances such as cysteine, thiomalic acid or glutathione in the
reduced state are supplied by subsequent treatment the block is removed and
morphogenesis continues.
SUMMARY
1. Chloroacetophenone (CAP), a specific inhibitor of SH groups, applied at
5 x 10~4 M for 15 min at the definitive primitive streak stage of chick embryos
causes abnormalities, predominantly in the nervous system.
2. The effects of CAP can be reversed by a subsequent treatment with thiomalic acid (5 x 10~4) or glutathione (1 x 10~5 M).
3. Equimolar concentration of serine (4 x 10~4), which is structurally similar
to cysteine except that SH of cysteine is replaced by OH in serine, fails to reverse
the effects of CAP.
PLATE 2
Fig. G. Embryo treated with CAP at definitive primitive streak followed by treatment with
serine. x36.
Fig. H. Transverse section through the above embryo.
Fig. I. Embryo treated with CAP at definitive primitive streak, x 36.
Fig. J. Embryo treated with CAP at definitive primitive streak followed by treatment with
glutathione. x36.
Fig. K. Embryo kept as absolute control without any treatment, x 36.
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L. MULHERKAR ET AL.
4. The results of the present work suggest that the reversal of the CAP
effects is brought about by SH groups.
RESUME
Reversibilite de Vaction de la chloracetophenone sur la morphogenese
d''embryons de poulet, au moyen de groupes sulphydriles
1. La chloracetophenone (CAP), inhibiteur specifique des groupes SH,
appliquee a des embryons de poulet pendant 15 minutes a la concentration de
5 x 10~4 M, au stade ligne primitive definitive, provoque des anomalies surtout
dans le systeme nerveux.
2. Les effets de la CAP peuvent etre annules par un traitement posterieur a
l'acide thiomalique (5 x 10~4 M) OU au glutathion (1 x 10~5 M).
3. Une concentration equimolaire de serine (4 x 10~4), laquelle possede une
structure similaire a la cysteine, excepte le fait que le SH de la cysteine est
remplace par OH dans la serine, ne peut annuler l'effet de la CAP.
4. Les resultats de ce travail suggerent l'hypothese que la reversibilite de
l'action de la CAP est provoquee par les groupes SH.
Two of us, S. S. Joshi and B. A. Diwan, are grateful to the Indian Council of Medical
Research for post-doctoral fellowships.
REFERENCES
BRACHET, J. (1950). Chemical Embryology. New York: Interscience Publishers.
LAKSHMI, M. S. (1962G). The effect of chloroacetophenone on chick embryos cultured in vitro.
J. Embryol. exp. Morph. 10, 373-82.
LAKSHMI, M. S. (19626). The effect of chloroacetophenone on the inducing capacity of
Hensen's node. /. Embryol. exp. Morph. 10, 383-8.
LAKSHMI, M. S. & MULHERKAR, L. (1963). Paper chromatographic study of the role of
sulphur-containing amino acids in the process of induction in the chick embryo. Experientia
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MULHERKAR, L., VASUDEVA RAO, K. & JOSHI, S. (1965). Studies on some aspects of the role
of sulfhydryl groups in morphogenesis. /. Embryol. exp. Morph. 14, 129-35.
MUZUMDAR, S. (1966). Sulfhydryl groups and embryonic induction. Ph.D. thesis, University
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NEW, D. A. T. (1955). A new technique for the cultivation of the chick embryos in vitro.
J. Embryol. exp. Morph. 3, 326-31.
RAPKINE, L. & BRACHET, J. (1951). Recherches sur le role des groupes sulfhydriles dans la
morphogenese. I. Bull. soc. chim. Biol. 33, 427.
WAHEED, M. A. & MULHERKAR, L. (1967). Studies on induction by SH-containing substances
in post-natal pieces of chick blastoderm. J. Embryol. exp. Morph. 17, 161-9.
{Manuscript received 18 July 1966)