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1979 Development of the Cervical Region of Chicken Embryos Studied via the Teratogenic Effects of Monocrotophos. Christina Irene Lusk Louisiana State University and Agricultural & Mechanical College
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Recommended Citation Lusk, Christina Irene, "Development of the Cervical Region of Chicken Embryos Studied via the Teratogenic Effects of Monocrotophos." (1979). LSU Historical Dissertations and Theses. 3341. https://digitalcommons.lsu.edu/gradschool_disstheses/3341
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University Microfilms International 300 N. ZEEB ROAD. ANN ARBOR. Ml 48106 18 BEDFORD ROW, LONDON WC1R 4EJ, ENGLAND 7921971 LUSK, CHRISTINA IRENE DEVELOPMENT OF THE CERVICAL REGION OF CHICKEN EMBRYOS STUDIED VIA THE TERATOGENIC EFFECTS OF M0N0CR0T0PH08. THE LOUISIANA STATE UNIVERSITY AND AGRICULTURAL AND MECHANICAL COL*# PH.D.# 1979
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International 300 N. ZEEB RD.. ANN ARBOR. Ml -18106 (313) 761-4700 DEVELOPMENT OF THE CERVICAL REGION OF
CHICKEN EMBRYOS STUDIED VIA THE
TERATOGENIC EFFECTS OF MONOCROTOPHOS
A Dissertation
Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Doctor of Philosophy
in
the Department of Zoology and Physiology
by Christina Irene Lusk B.S. Mississippi State University, 1970 M.S. Mississippi State University, 1972 May 1979 Acknowledgement
I wish to express my gratitude to my major professor, Dr. Mary
L. Grodner, who supported and helped me in any way possible in this project. I especially wish to thank her for help with the SEM work.
I wish to thank W. L. Steffens for technical assistance with photography and TEM, and for training in the use of the Hitachi
HU-11 A. Thanks go to my friends Barbara Bel isle and Chris Smith, who helped print plates, and to Linda Floyd, who assisted in typing the final draft of this paper.
I wish to thank Dr. Bill Johnson and the LSU Poultry Science
Department for providing the fertile White Leghorn eggs necessary for this project.
I also want to thank my committee, Dr. Ned Lambremont, Dr. Jerry
Graves, Dr. Bill Byrd, Dr. Henry Werner, and Dr. J. P. Woodring for their support and advice. Table of Contents
Title Page i
Acknowl edgement i i
Table of Contents ...... iii
List of Tables iv
List of Figures v
Abstract xiii
Introduction 1
Literature Review ...... 3
Materials and Methods ...... 14
Results 19
Discussion 37
Conclusion 47
Literature Cited 50
Plates 55
Vita 136
iii List of Tables
Table 1 Comparison of body parameters for 2 and 3 day treatme.nts ...... 20
Table 2 Stage of. development and per cent abnormal embryos at various hours after treatment ...... 23
Table 3 Histochemical test for acetylcholinesterase of tissue from the cervical region of stage 19+ chicken embryos treated in vitro. with in h ib ito rs ...... 33
Table 4 Histochemical test for acetylcholinesterase after in ovo treatments of stage 19 - 20 chicken embryos ...... 34
iv List of Figures
Fig. 1 Control, treatment before incubation ...... 56
Fig. 2-3 Embryo treated with monocrotophos before incubation ...... 56
Fig. 4 Control embryo, treatment at 1 day of incubation ...... 58
Fig. 5 Embryo treated with monocrotophos at 1 day of incubation ...... 58
Fig. 6 Control embryo, treatment at 2 days of incubation ...... 60
Fig. 7 Embryo treated with monocrotophos at 2 days of incubation ...... 60
Fig. 8 Control embryo, treatment at 3 days of incubation ...... 62
Fig. 9 Embryo treated with monocrotophos at 3 days of incubation ...... 62
Fig. 10 Control embryo, treatment at 4 days of incubation ...... 64
Fig. 11-12 Embryo treated with monocrotophos at 4 days of incubation ...... 66
Fig. 13 Control embryo, treatment at 5 days of incubation ...... 68
Fig. 14 Embryo treated with monocrotophos at 5 days of incubation ...... 68
Fig. 15 Control embryo, 3 hours after treatment at 3 days of incubation, SEM ...... 70
Fig. 16-18 Embryo 3 hours after treatment with monocrotophos at 3 days of incubation, SEM ...... 70
v vi
Fig. 19 Control embryo, 3 hours after treatment at 3 days of incubation ...... 72
Fig. 20 Embryo 3 hours after treatment with mono crotophos at 3 days of incubation ...... 72
Fig. 21 Embryo 3 hours after treatment with mono crotophos at 3 days of incubation ...... 74
Fig. 22 Control embryo, 3 hours after treatment at 3 days of incubation, 1 urn c.x ...... 74
Fiq. 23 Embryo'3 hours after treatment with mono crotophos at 3 days of incubation, 1 urn c.x. . . . . 74
Fig. 24 Marginal layer of ventral edge of neural tube, control embryo, 3 hours after treat ment at 3 days of incubation, TEM ...... 76
Fig. 25 Marginal layer of ventral edge of neural tube, embryo 3 hours after treatment with monocrotophos at 3 days of incubation, TEM ...... 76
Fig. 26 Notochord, control embryo, 3 hours after treatment at 3 days of incubation, TEM ...... 76
Fig. 27 Notochord, embryo 3 hours after treatment with monocrotophos a t.3 days of incubation, TEM...... 76
Fig. 28 Hyotome, control embryo, 3 hours after treatment treatment at 3 days of incubation, TEM ...... 78
Fig. 29 Ityotome, embryo 3.hours after treatment- with monocrotophos at 3 days of incubation, TEM...... 78
Fig. 30 Sclerotome, control embryo, 3 hours after treatment at 3 days of incubation, TEM ...... 78
Fig. 31 Sclerotome, embryo 3 hours after treatment with monocrotophos at 3 days of incubation, TEM...... 78
Fig. 32 Control embryo, 6 hours after treatment at 3 days of incubation, SEM ...... 80
Fig. 33 Embryo 6 hours after treatment with mono crotophos at 3 days of incubation, S E M ...... 80
Fig. 34-35 Control embryo, 6 hours after treatment with monocrotophos at 3 days of incubation ...... 80 Embryo 6 hours after treatment with mono crotophos at 3 days of incubation .... 82
Control embryo, 6 hours after treatment at 3 days of incubation, 1 urn c.x ...... 84
Embryo 6 hours after treatment with mono crotophos at 3 days of incubation, 1 urn c.x 84
Marginal layer of ventral edge of neural tube, control embryo, 6 hours after treat ment at 3 days of incubation, TEM .... 84
Marginal layer of ventral edge of neural tube, embryo 6 hours after treatment with monocrotophos at 3 days of incubation, TEM 84
Notochord, control embryo, 6 hours after treatment at 3 days of incubation, TEM . . 86
Notochord, embryo 6 hours after treatment with monocrotophos at 3 days of incubation, TEM ...... 86
Myotome, control embryo, 6 hours after treatment at 3 days of incubation, TEM . . 88
Myotome, embryo 6 hours after treatment with monocrotophos at 3 days of incubation; TEM ...... 88
Sclerotome, control embryo, 6 hours after treatment at 3 days of incubation, TEM . . 88
Sclerotome, embryo 6 hours after treatment with monocrotophos at 3 days incubation, TEM ...... 88
Control embryo, 9 hours after treatment at 3 days of incubation, SEM ...... 90
Embryo 9 hours after treatment with mono crotophos at 3 days of incubation, SEM . . 90
Control embryo, 9 hours after treatment at 3 days of incubation ...... 90
Embryo 9 hours after treatment with mono crotophos at 3 days of incubation .... 92 Control embryo, 9 hours after treatment at 3 days of incubation, 1 urn c.x ...... 94 vi i i
Fig. 55 Embryo 9 hours after treatment with mono crotophos at 3 days of incubation, 1 urn c.x. .
Fig. 56 Marginal layer of ventral edge of neural . tube, control embryo, 9 hours after treat ment at 3 days of incubation, TEM ......
Fig. 57 Marginal layer of ventral edge of neural tube, embryo 9 hours after treatment with monocrotophos at 3 days of incubation, TEM ......
Fig. 58 Notochord, control embryo, 9 hours after treatment at 3 days of incubation, TEM ....
Fig. 59 Notochord, embryo 9 hours after treatment with monocrotophos at 3 days of incubation, TEM ......
Fig. 60 Myotome, control embryo, 9 hours after treatment at 3 days of incubation, TEM ...... 96
Fig. 61 Myotome, embryo 9 hours after treatment with monocrotophos at 3 days of incubation, TEM ......
Fig. 62 Sclerotome, control embryo, 9 hours after treatment at 3 days of incubation, TEM ...... 98
Fig. 63 Sclerotome, embryo 9 hours after treatment with monocrotophos at 3 days of incubation, TEM ......
Fig. 64 Control embryo, 12 hours after treatment at 3 days of incubation, SEM ...... 98
Fig. 65 Embryo 12 hours after treatment with mono crotophos at 3 days of incubation, SEM ...... 98
Fig. 66 Control embryo, 12 hours after treatment at 3 days of incubation ...... , . . 100
Fig. 67-68 Embryo 12 hours after treatment with mono crotophos at 3 days of incubation ...... JOG
Fig. 69 Control embryo, 12 hours after treatment at 3 days of incubation, lum c.x ...... 102
Fig. 70 Embryo 12 hours after treatment with mono crotophos at 3 days of incubation, 1 urn c.x. . 102 Fig. 71 Marginal layer of ventral edge of neural tube, control embryo, 12 hours after treat ment at 3 days of incubation, TEM ...... 102
Fig. 72 Marginal layer of ventral edge of neural tube, embryo 12 hours after treatment with monocrotophos at 3 days of incubation, TEM ...... 102
Fig. 73 Myotome, control embryo, 12 hours after treatment at 3 days of incubation, TEM ...... 104 Fig. 74 Myotome, embryo 12 hours after treatment with monocrotophos at 3 days of incubation, T E M ...... 104
Fig. 75 Notochord, control embryo, 12 hours after treatment at 3 days of incubation, TEM ...... 1C4
Fig. 76 Notochord, embryo 12 hours after treatment with monocrotophos at 3 days of incubation, T E M ...... 104
Fig. 77 Sclerotome, embryo 12 hours after treatment with monocrotophos at 3 days of incubation, TEM ...... 106
Fig. 78 Sclerotome, control embryo, 12 hours after treatment at 3 days of incubation, TEM ...... 106
Fig. 79 Control embryo, 1 day after treatment at 3 days of incubation, SEM ...... 106
Fig. 80 Embryo 1 day after treatment with monocro tophos at 3 days of incubation, SEM ...... 106
Fig. 81 Control embryo, 1 day after treatment at 3 days of incubation ...... 108
Fig. 82-83 Embryo 1 day after treatment with monocro tophos at 3 days of incubation ...... 108
Fig. 84 Control embryo, 1 day after treatment at 3 days of incubation, 1 urn c.x ...... 110
Fig. 85 Embryo 1 day after treatment with monocro tophos at 3 days of incubation, 1 urn c.x...... 110
Fig. 86 Marginal layer of ventral edge of neural tube, control embryo, 1 day after treat ment at 3 days of incubation, TEM ...... 110 X
Fig. 87 Marginal layer of ventral edge of neural tube, embryo 1 day after treatment with monocrotophos at 3 days of incubation,TEM ...... 11°
Fig. 88 Notochord, control embryo, 1 day after treatment at 3 days of incubation, TEM ...... 112
Fig. 89 Notochord, embryo 1 day after treatment with monocrotophos at 3 days of incubation, TEM ...... 11-
Fig. 90 Myotome, control embryo 1 day after treat ment at 3 days of incubation, TEM ...... 114
Fig. 91 Myotome, embryo 1 day after treatment with monocrotophos at 3 days of incubation, TEM ...... 114
Fig. 92 Control embryo, lh days after treatment at 3 days of incubation, SEM ...... 114
Fig. 93 Embryo lh days after treatment with mono crotophos at 3 days of incubation, SEM ...... 114
Fig. 94 Control embryo, lh days after treatment at 3 days of incubation ...... 116
Fig. 95 Embryo lh days after treatment with mono crotophos at 3 days of in c u b a tio n ...... 116
Fig. 96 Control embryo, lh days after treatment at 3 days of incubation, 1 urn c.x ...... 116
Fig. 97 Embryo lh days after treatment with mono crotophos at 3 days of in c u b a tio n ...... 116
Fig. 98 Notochord, control embryo, lh days after treatment with monocrotophos at 3 days of incubation, TEM ...... 118
Fig. 99 Notochord, embryo lh days after treatment with monocrotophos at 3 days of incubation, TEM ...... 118
Fig. 100 Myotome, control embryo, lh days after treatment at 3 days of incubation, TEM ...... 118
Fig. 101 Myotome, embryo lh days after treatment with monocrotophos at 3 days of incubation, TEM...... 118 XI
Fig. 102 Control embryo, 2 days after treatment at 3 days of incubation, SEM ...... 120
Fig. 103 Embryo 2 days after treatment with monocro tophos at 3 days of incubation, SEM ...... 120
Fig. 104 Control embryo, 2 days after treatment with monocrotophos at 3 days of incubation ...... 120
Fig. 105 Embryo 2 days after treatment with monocro tophos at 3 days of incubation ...... 120
Fig. 106 Control embryo, 2h days after treatment at 3 days of incubation ...... 122
Fig. 107 Embryo 2h days after treatment with monocro tophos at 3 days of incubation ...... 122
Fig. 108 Control embryo, 3 days after treatment at 3 days of incubation ...... 122
Fig. 109 Embryo 3 days after treatment with monocro tophos at 3 days of incubation ...... 122
Fig. 110 Control embryo, 5 days after treatment at 3 days of incubation ...... 124
Fig. 111 Embryo 5 days after treatment with monocro tophos at 3 days of incubation ...... 124
Fig. 112 Control in vitro, AChE stain, ACH substrate .... 126
Fig. 113 Control in vitro, AChE stain, no substrate ...... 125
Fig. 114 Eserine treated in vitro, AChE stain, ACH substrate ...... 126
Fig. 115 iosOMPA treated in vitro, AChE stain, ACH substrate ...... 1.26
Fig. 116 284C51 treated in vitro, AChE stain, ACH substrate ...... 126
Fig. 117 Control in vitro, AChE stain, BuThCh substrate ...... 123
Fig. 118 isoOMPA treated in vitro, AChE stain, BuThCh substrate ...... 128
Fig. 119 284C51 treated in vitro, AChE stain, BuThCh substrate ...... 128 xii
Ffg. 120 Control in ovo, AChE s t a i n ...... 128
Fig. 121 isoOMPA treated in ovo, AChE s t a i n ...... 128
Fig. 122 284C51 treated in ovo, AChE s t a i n ...... 128
Fig. 123 Monocrotophos treated in ovo, AChE s t a i n ...... 130
Fig. 124 Monocrotophos treated, cartilage stain ...... 130
Fig. 125 isoOMPA treated, cartilage stain ...... 130
Fig. 126 Eserine treated, cartilage stain ...... 130
Fig. 127-128 284C51 treated, cartilage stain ...... 130
Fig. 129 Control embryo, cartilage s t a i n ...... 130-
Fig. 130 Control embryo, cartilage stain ...... 132
Fig. 131-132 Monocrotophos treated, cartilage stain ...... 132
Fig. 133-135 Embryos treated with both monocrotophos and 2-PAM at 3 days of incubation, cartilage stain ...... 132
Fig. 136-137 Embryos treated with 2-PAM at 2 days of incubation, then with both monocrotophos . and 2-PAM at 3 days of incubation, cartilage s t a i n ...... 134
Fig. 138-139 Embryo treated with both 2-PAM and monocro tophos at 3 days of incubation then with 2-PAM at 4 days of incubation, cartilage s t a i n ...... 134
Fig. 140 Monocrotophos plus 2-PAM treated in ovo, AChE s t a i n...... 134
Fig. 141 Control in ovo, AChE s t a i n ...... 134 Abstract
The teratogenic effects of an organophosphate insecticide,
monocrotophos, were studied during the embryonic development of the
neural tube, notochord, myotome and vertebrae of the cervical region
of White Leghorn chickens. Embryos 0 to 5 days old were treated
once with 100 u_g of monocrotophos and fixed at 6 days of incubation.
The resulting cervical defects became increasingly severe with
treatments at 0 to 2 days, were most severe when treated at 3 days
of incubation, then decreased in severity when treated on the 4th
or 5th day. The defects consisted of fused vertebral centra and
neural arches, a warped notochord, and poorly developed muscle.
Light microscopic and electron microscopic studies were made
of the development of the defects with treatment at 3 days of
incubation (stage 20). The embryos were first observed at 3 hours
after treatment at which time 39% of the embryos were abnormal
(external observation). The abnormalities consisted of a myotome that
was shorter than normal and a distortion of the areas lateral to the
n\yotome. Indications of the same abnormalities were observed
externally for all treatment periods studied using SEM.
The arrangement of the actin and myosin filaments of the myofibrils
in the treated embryos was disorganized by 3 hours after treatment. No myofibrils were observed by 6 hours after treatment. The myofibrils
started to recover from the effects of the insecticide by 24 hours xiii xiv but were still fewer in number in the treated than in the controls by
36 hours after treatment.
The notochord developed a dorsoventrally warped appearance, which became lateral and increased in severity as development continued. The vertebral centra developing around the notochord were wedge-shaped and fused, not rectangular as in the controls. The neural arches were also fused. The first 8 to 10 cervical vertebrae had developed as a solid mass of cartilage by 5 days after treatment. This region was one-half the length of a normal cervical region.
An histochemical stain for acetylcholinesterase (AChE) showed this enzyme to be present in the myotome and the neural tube, but not in the notochord of stage 20 embryos. Those compounds tested which inhibited AChE in ovo shortly after treatment at approximately stage 20 also caused the cervical defects when the embryos were observed at stage 29 (6 days of incubation). Counter treatment with pyridine-2- aldoxime methiodide (2-PAM), an AChE reactivator, corrected some but not all of the defects caused by monocrotophos. Some embryos that were treated with both 2-PAM and monocrotophos looked like normal ones, while others had fused and curved cervical vertebrae. All embryos treated with 2-PAM had an increase in neck length over those of the monocrotophos treated embryos.
It was concluded that inhibition of AChE interfered with muscle development. This in some way caused a shortening or compression of the cervical region. With less than normal room to elongate, the notochord was forced into a curved shape. The vertebrae developed wedge-shaped to fit around these curves, and fused shortened vertebrae were the result. Introduction
Many organophosphate and methyl carbamate insecticides have been found to be teratogenic when injected into bird eggs (Kera and Bedok
1967; Roger 1967; Meiniel et al_. 1970; Strudel 1971; Meiniel 1975b;
Proctor and Casida 1975; Moscioni et_ al_. 1977). The resulting defects are a twisted reduced neck, feather abnormalities, size reduction, beak defects, and limb defects. One to all of the defects are present when a specific insecticide is used.
The notochord, neural tube, somites and other structures in the cervical region influence each other during development. To learn more about normal development, and the interactions of these structures, abnormal development was studied. Monocrotophos was used as the terato genic agent in these studies to disrupt normal development. Monocroto phos (dimethyl phosphate of 3-hydroxy-N.-methyl ci£ - crotonamide)
(Azodrin ( ) f t is a vinyl organophosphate insecticide manufactured by
Shell Chemical Company. The chemical structure is as follows:
0 (CH,0),P-H ,H c = c H3c ' jjN H C H g
Organophosphate insecticides are strong inhibitors of the enzyme acetylcholinesterase, and as such disrupt normal nerve impulse transmission. Bull and Lindquist (1964) first reported monocrotophos as a metabolite of dicrotophos (Bidrin®). It is a minor metabolite in cotton and a major metabolite in rats.
The following questions were asked in this study in order to determine the relationships and interactions of the different components of the cervical region during development.
1. What are the gross effects of monocrotophos treatment?
2. Is there a difference in the resulting defect when the
embryos are treated at different times? If so, when are the
embryos most sensitive?
3. How does the defect develop at the most sensitive time
period?
4. Does monocrotophos inhibit acetylcholinesterase in the
early embryo?
5. Can the defect be corrected using agents that reactivate
acetylcholi nesterase?
6. What is being disrupted in early development to cause
this defect? Literature Review
Vinyl phosphate pesticides, such-as dicrotophos or monocrotophos,
injected in chicken eggs before incubation caused numerous teratogenic
effects when the embryos were observed near term. Among these effects were abnormal feathering, parrot beak, shortened and twisted neck,
and edema (Proctor and Casida 1975; Roger 1967; Roger et al_. 1964;
1969).
The cis-crotonamide isomer of dicrotophos was found to be the most active, being teratogenic at a dose of 30 .ug/egg and higher.
The teratogenic effects become more severe with increasing dosage.
Dicrotophos causes the above defects when injected during the first
6 days of incubation, but not after 9 days. Treatment at 4 days of incubation was found to cause the severest defects (Roger 1967;
Roger ertal_. 1964; 1969).
White Leghorn embryos observed in the egg were deformed 3 hours after monocrotophos treatment at stage 18 and 1 hour after treatment at stage 20. The defect, in the neck region between the aortic arches and wing buds, consisted of an abnormal bending. Monocrotophos residues in developing eggs had decreased by one half by 48 hours but remained at this level for the next 48 hours (Schom and Abott 1977).
Nicotinic acid analogs or precursors such as nicotinamide, NAD,
NADH, NADP, or NADPH alleviated the symptoms of the dicrotophos syndrome except for the cervical defects (Proctor and Casida 1975; 3 Roger 1967; Roger et^a]_. 1964; 1969). There seemed to be a correlation between NAD levels and the teratogenic effects of certain insecticides.
The inhibition of acetylcholinesterase (AChE) and other esterases by dicrotophos does not correlate directly with the degree of terato- genesis (Proctor al_. 1976; Upshall et^al_. 1968). Yolk sac membrane esterases were tested using a teratogenic (dicrotophos) and non- teratogenic (EPN) organophosphate. Using gel electrophoresis, esterase bands were found that were inhibited by dicrotophos, but not by EPN (Flockhart and Casida 1972). A more refined technique later showed that there was no correlation between inhibition of a specific esterase band and organophosphate teratogenesis (Proctor et^al_. 1976).
Malathion, injected into chicken eggs at 3.99 or 6.42 mg/egg from day 4 to 12 of incubation, caused shortened legs, curled toes, parrot beak^ lack of feathers, and general reduction in body size, but no cervical defects (Greenberg and LaHam 1969). Tryptophan, quinolinic acid, nicotinamide, and nicotinic acid were tested as counter agents. Tryptophan was the best agent with no malformations and normal size after treatment. Five to 10 per cent malformations and growth retardation remained when the others were used (Greenberg and LaHam 1970). The authors concluded that the reduction in embryo tryptophan correlated with the defects caused by malathion. Because tryptophan is necessary for NAD synthesis, the results obtained by
Greenberg and LaHam agreed with those obtained by Proctor and Casida
(1975), Roger (1967), and Roger et^al_. (1964 ;1969).
Moscioni et_al_. (1977) reported a good correlation between decreased NAD levels at 12 days of incubation and the severity of micromelia and abnormal feathering at 19 days using 26 organophosphate and carbamate insecticides. Kynurenine formamidase, an enzyme in the pathway that liberates tryptophan from the yolk, was inhibited by these same compounds. This inhibition caused reduced NAD synthesis due to lowered amounts of tryptophan available. Highly teratogenic compounds lowered kynurenine formamidase activity more than 75%
1 hour after treatment or sustained inhibition of 40% for 24 hours after treatment.
Strudel (1971) and Strudel and Gateau (1971) injected chicken eggs with nicotine sulfate at 2 or 4 days of incubation at dose levels of 1 to 5 mg/egg, causing deformities of the cervical region. The notochord curved laterally by 8 hours after treatment in 44% of the embryos. The neural tube and somites were normal. The notochord was warped more, with the other areas normal by 12 hours after treatment in 65% of the embryos. The neural tube as well as the notochord had a sinusoidal appearance by 24 hours. The defect was located between the otic capsule and the eleventh vertebrae with 84% of the embryos affected. The third to eleventh vertebrae were shortened in an anterior - posterior direction by 36 hours, affecting 98% of the embryos. There was a gross curvature of the neural tube and notochord and compression of the organs in this region. All the previously described signs were present and more accentuated by 48 hours with all the embryos affected. The authors concluded that the deformation of the notochord caused the deformation of the neural tube.
Coturnix quail, treated before incubation by dipping in an acetone solution of parathion (2% active ingredient), were observed grossly and microscopically at 12 days of incubation. Spinal defects, especially in the cervical region, were the main abnormalies observed. The neck length was reduced and the neck twisted, resulting in the head lying on the right side of the embryo. Multiple curvatures of the spine in the saggital plane affecting the cervical, thorasic, lumbar sacral and caudal regions were observed, resulting in a S-shaped spine. These curves caused a shortening in the embryonic body length.
The cervical curvature was the most pronounced, with fusion of the neural arches of the first 7 to 8 vertebrae. The eighth was free and served as the pivot for the twisting towards the right. The occipital condyle was fused with the odontoid process of the axis
(Meiniel 1970; 1975b; 1977a; Meiniel et al_. 1970).
Treatment by injection of parathion in olive oil (0.25 to 1 mg) resulted in the same defects as dipping. When chicken eggs were treated at 0,1, or 2 days of incubation, no defects were found until the fourth day of incubation. At this time the notochord appeared warped in a lateral direction creating a sinusoidal effect. The symmetry of the developing vertebrae and somites was disrupted. A curvature of the cervical region had developed by 4% days of incubation
(stage 24 to 26) (Meiniel 1973a;1973b;1975b;1977a). Histologically by'this stage a concave curve affected the notochord and neural tube at the level of the eighth to tenth vertebrae. The notochord curve became more accentuated with time, with the neural tube following the notochord's path. The somites were segmented between 115 and 120 hours, but with irregular spacings, especially in the curved region.
Chondrification proceeded normally, but with collagen and glycosamino- glycans being laid down in the intervertebral spaces. This occurred between the fifth and sixth days of incubation. All of the chicken embryos treated on 5, 6, 7, 8, or 10 days of incubation with 0.25 to 0.5 mg parathion and removed 3 or 4 days after treatment showed cervical deformations. Grossly these appeared the same as those of embryos treated before incubation. The neck was twisted and shortened with the head lying on the right side of the body. The vertebrae in these later treatments were rarely fused, but reduction of the intervertebral spaces was seen. Abnormal muscle development was demonstrated by the lack of organization of the muscle myofibrils (Meiniel 1973a; 1973b; 1974a; 1975b; 1977a).
Quail eggs, treated with parathion, received single or multiple doses of 1 to 2 mg pralidoxime, a cholinesterase reactivator.
Parathion treatment alone resulted in the neck defect. Parathion plus pralidoxime treatment at 4,5,and 6 days of incubation resulted in no fusion of the neural arches (Meiniel 1974b; 1974c; 1975b).
Quail eggs were treated before incubation with 250 £g dicrotophos only and with 1 mg pralidoxime at both 3 and 5 days. Dicrotophos treated embryos had defects as described earlier for this pesticide.
The neck defects were corrected in embryos counter treated with pralidoxime with some increase in body length, but the other anomalies were still present (Meiniel 1975a).
Parathion treated quail eggs (100 ug, 0 day) were counter treated with 1 to 2 m§ of one of the following agents at 3 days of incubation; pyridine-2-aldoxime methyl methane sulfonate (P2S); pyridine-2-aldoxime methiodide (2-PAM); N.-N.-tri methyl, 1,1,3-bis pyridinium-4-aldoxime bromide (TM3-4); atropine; D-L-tryptophan; dimethyl sulfoxide (DMSO); monoisonitrosoacetone (MINA); or diacetylmonoxime (DAM). The use of P2S, 2-PAM, and TKB-4 reduced the 8 severity of the defects similar to the action of pralidoxime. The other compounds tested had no effect on reversing the defects.
Meiniel (1976a; 1976b) concluded that a pyridinium nucleus, quaternary nitrogen, and aldoxime function were necessary to counteract the effects of parathion.
Quail eggs were treated before incubation with either 75 u^ parathion or 250 jig dicrotophos alone or counter treated with l' mg nicotinamide or 2 mg pralidoxime at 3 days. The expected syndromes resulted when the eggs were treated with the insecticide alone. A normal looking embryo resulted with parathion plus pralidoxime treatment. The parathion syndrome was present with parathion plus nicotinamide treatment. Dicrotophos plus pralidoxime corrected spinal defects, while beak, limb, and feather defects remained.
Dicrotophos plus nicotinamide corrected beak, limb, and feather defects while the spinal ones remained. These results demonstrated a dual mode of teratogenic action with dicrotophos (Meiniel 1976a;
1976c).
Meiniel (1977b) injected quail eggs at 3 days of incubation with either 1 mg malathion, 250 u$ dicrotophos, or 1 to 100 u,g parathion. These embryos were examined at 8 to 11 days for correlation between axial abnormalities and AChE inhibition. AChE was only slightly inhibited and no cervical defects resulted with malathion treatment. AChE activity was greatly reduced, and axial deformities were present with dicrotophos treatment. AChE activity was slightly reduced, but the embryos had a normal external appearance when treated with 1 jug parathion. Increasing amounts of parathion up to 10 u.g increased the degree of deformity, with more vertebral fusion, and increased the degree of AChE inhibition.
Meiniel concluded that there was a positive correlation between
AChE inhibition and the observed cervical defects. This is opposite the conclusion of Proctor et al_. (1976) and Upshall et aK (1968).
Treatment of 3 or 4 day chicken embryos with carbachol chloride, decamethoniurn iodide, neostigmine bromide (Landauer 1975;
Meiniel 1978), succinylcholine chloride, trimethyl phenyl ammonium iodide, tetramethylammonium chloride, pyridostigmine bromide
(Landauer 1975), nitrostigmine, demecarium bromide, or physostigmine
(eserine) (Meiniel 1978) resulted in a shortened cervical region.
These compounds are either antagonists of acetylcholine which depolarize postsynaptic junctions, or anticholinesterase agents.
Treatment with gall amine (Landauer 1975; Meiniel 1978), benzo- qui.nonium (Landauer 1975) or hexamethonium (Meiniel 1978) countered the teratogenic agents, resulting in normal looking embryos (external view only). The incidence of the defect caused by carbachol or neostigmine was greatly reduced by 2-PAM and P2S treatments
(Landauer 1977).
Several authors (Drachman and Sokol off 1966; Murry and
Drachman 1969; Sullivan 1966) have demonstrated that movement plays an important role in joint formation. Skeletal muscle contractions have been observed in chicken embryos as early as 3k to 4 days of incubation. Paralysis of these muscles produced teratogenic effects in the skeletal system. Decamethoniurn, an inhibitor of muscle contraction, was used to treat embryos during early development.
The resulting embryos were slightly retarded in growth. The head was deflected to the right with a sharp 90 degree bend in the neck. 10
The vertebral column was rotated on its axis in a counter clockwise
direction. The vertebral column had buckled into an S-shaped curve
due to anterior - posterior compression of the body. Fusion between cartilagenous vertebrae occurred, being most noticeable in the neural arch region. There was cartilagenous fusion between the atlas and the base of the skull (Sullivan 1966).
Embryo paralysis caused joint cavities to fail to develop in chicken embryos. Joint interzones were first occupied by a vascular connective tissue and later by compact fibrous, cartilagenous, or bony tissue depending on the composition of the elements being fused
(Drachman and Sokol off 1966; Murry and Drachman 1969).
Removal of the notochord in young embryos caused an unsegmented ventral ^cartilagenous mass to develop instead of the vertebral centra.
The dorsal region had normal neural arches. Removal of the neural tube caused either absence of or abnormal neural arches to form
(Strudel 1955a; 1955b; 1967).
Acetylcholinesterase (AChE) is usually mentioned in connection with cholinergic nerves, neuromuscular and neurotendenous j'unctions.
Numerous authors have reported AChE in non-neural locations. Bishop et al_. (1976) reported the presence of a cholinergic system in both bull and human sperm. All parts of the sperm contained AChE, but the tail contained the highest amounts. It was postulated that this system played an important role in controlling sperm motility.
Sastry et al_. (1976) demonstrated that the human placenta contains AChE-and acetylcholine (ACh) even though it lacks innervation.
The ACh was located mainly in the syncytiotrophoblast with the highest 11 levels in the second trimester. The placental cholinergic system may
play a role in regulation of the transport of nutrients and chemicals across the syncytiotrophoblast and thereby regulate fetal growth.
During the second to sixth months of pregnancy, i t is difficult for chemicals to cross the placental barrier. This is the time that the cholinergic system is the most active. During the other times in pregnancy, chemicals can cross the barrier. This coincided with the time of lesser cholinergic activity.
AChE is located internally in rat liver cells in smooth and rough endoplasmic reticulum, in golgi apparatus, in the nuclear membrane, and between cells at tight junctions (Satler et al_. 1974).
Hepatic tyrosine transaminase was increased by stimulation of the vagus and by cholinergic drugs. This was antagonized by drugs which block protein synthesis. A cholinergic mechanism was affecting protein synthesis in this case. The subcellular localization of
AChE activity indicated the sites of acetylcholine interaction with protein synthesis. ACh could influence the release of RNA to the cytoplasm as well as the ribosomes (Black and Reis 1971).
Bartos and Glinos (1976) demonstrated that AChE was localized on cell membranes and was released into the medium in cultured
WRL-10A fibroblasts, a subline of L-929 mouse fibroblasts. Their work also showed that AChE activity increased 100 fold when contact inhibition occurred.
AChE activity occurres in embryonic skeletal muscle cells from chicken embryos cultured without nerves. The activity is in the sarcolemma, in subsurface vacuoles, in sacrotubular membranes, 12
in the nuclear envelope, and outside the cells. The nucleoplasm, mitochondria and myofilaments contain no activity (Sawyer et al_. 1976).
The AChE system may be involved in events concerning spontaneous contraction of cardiac muscle according to Taylor (1976). Embryonic chicken heart exhibits cholinergic activity from stage 3 (12 hours) to 4 days after hatching. During the first 14 days of incubation specific acetylcholinesterase activity is present. However, after 14 days of incubation only nonspecific or pseudocholinesterase occurs. The activity is located in the golgi apparatus, sacroplasm reticulum, rough endoplasmic reticulum (RER), T-tubule system, nuclear membrane, and sacrolemma. The exact location varies as to the stage of development. AChE activity occurs in the n\yofilaments as soon as they appear (stage 10). This activity increases with development.
Drews (1975) studied AChE during embryonic development in chickens.
Abundant AChE was found in tissues independent of the nervous system.
Every organ studied ran through a pliase of AChE activity during differentiation. After the organ structure had been developed, the AChE activity decreased. Histochemical methods demonstrated that the cells involved in morphogenic movements contained AChE. Drews suggested that AChE might be connected with an actin - myosin system used for cellular movements. Sclerotomic cells migrating from the somites show a positive reaction to AChE stains. They become negative when migration stops and they orient near the notochord.
These cells again become positive during cartilage development. The developing myotome, ventral ganglia, ventral horn of the spinal cord, 13 apical ectodermal ridge, mesonephros, and lining of blood vessels are positive at stage 20 (about 3 days of incubation). AChE activity was found during the differentiation of cartilage with maximum levels at stage 24, and during muscle and nerve development of all stages. Materials and Methods
General methods
Fertile White Leghorn eggs were obtained from the LSU Poultry
Farm on Perkins Road. Eggs were incubated at 38 to 39.5°C (still air)
for the desired time before and after treatment. All injections were made into the yolk using a 50 £l Hamilton syringe. Boiled distilled water was used as the solvent in all cases. Control embryos were
injected with the same volume of water as treated embryos at the same treatment period.
The monocrotophos used in this study (analytical standard,
Shell Chemical Company) was obtained from the Entomology Department,
LSU.
Gross effects of monocrotophos
The eggs were injected with 50 _ug of monocrotophos at 2 or
3 days of incubation. The eggs were returned to the incubator and incubated a total of 17 days. The embryos were then removed from the eggs and fixed in 10% formaldehyde. The lengths of the body, legs, wings, neck, head, beak, and middle toe were measured.
Defect appearance versus day of treatment
Fertile eggs were injected with 100 ucj of monocrotophos before incubation, or at 1, 2, 3, 4 or 5 days of incubation. All embryos
14 15 were removed at 6 days of incubation and fixed in Bouin's fixative.
Slides for examination by light microscope were prepared (Humason
1972),. and the sections were stained using a modification of
Mallory's trichrome stain (Everett and Miller 1973).
Development of defects with treatment at J3 days of incubation
The eggs were incubated for 3 days then injected with 100 jjg of monocrotophos. The eggs were returned to the incubator until the required time.
For light level studies the control and treated embryos were removed 3, 6, 9 and 12 hours after treatment, and every 12 hours thereafter for a total of 3 days following treatment, and on 5 days after treatment. The embryos were fixed in Bouin's fixative. They were either stained in toto in alum cochineal (Guyer 1953), embedded and sectioned, or embedded in paraplast, sectioned at 10 urn, and stained with a modification of Mallory's trichrome stain (Everett and Miller 1973).
Control and treated embryos were removed 3, 6, 9, 12, 24, and
36 hours after treatment for transmission electron microscopic (TEM) studies. The embryos were fixed in 5% glutaraldehyde in 0.1M cacodylate buffer pH 7.2 for 1 hour, post fixed in 1% osmium tetroxide in 0.1M cacodylate buffer, pH 7.2, stained in toto in 0.53> uranyl acetate overnight, dehydrated using acidified
2,2-dimetho*ypropane (DNP), and embedded in epon-araldite (Dawes 1971).
Cross sections of the cervical region were cut at 1 micrometer and stained with toluidine blue (Dawes 1971). Pale gold to silver sections were cut from the desired areas using a MTB-2 ultramicrotone. 16
Grids were stained with uranyl acetate and Reynold's lead citrate.
Grids were observed using an Hitachi HU-11A Transmission Electron
Microscope.
Embryos were removed on the same schedule for scanning electron microscopic (SEN) studies as used for TEM studies. The embryos were fixed in FAA (10% formalin, 85% ethanol, 5% glacial acetic acid), rapidly dehydrated in acidified DKP and dried using a Denton critical point drying apparatus. The embryos were mounted on stubs using
Television Tube Koat® . They were coated with 200A of gold-palladium using a Hummer sputter-coater. Specimens were observed using an
Hitachi S-500 Scanning Electron Microscope.
Histochemical stain for acetylcholinesterase
The following chemicals were obtained from Si p a Chemical
Company: acetyl thiocholine iodide, butyryl thiocholine iodide, eserine sulfate, tetraisopropylpyrophosphoramide, and 1,5-bis
(4-allyldimethylammoni umphenyl)-pentane-3-1 di bromi de.
Embryos were stained for acetylcholinesterase (AChE) to determine the effects of monocrotophos on this enzyme. Inhibition by eserine indicates the general enzyme class cholinesterase
(Pearse 1972). False or pseudocholinesterase is specific for the substrate butyryl thiocholine iodide (BuThCh) and in inhibited by tetraisopropylpyrophosphoramide (isoOMPA). True or acetylcholinesterase is specific for the substrate acetyl thiocholine iodide and is inhibited by 1,5-bis (4-allyldimethylammoniumphenyl)-pentane-3-l dibromide
(previously identified in the literature as 284C51) (Pearse 1972). 17
Two published methods for AChE localization were attempted. The
method of Tsuji (1974) resulted in nonspecific stain. A modification
of the method of Karnovsky and Roots (1964) resulted in a positive
reaction with little or no nonspecific reaction. The embryos were
fixed in 4% formalin + CaCl 2 for 1 hour, then stored in 0.1M acetate
buffer overnight. The neck region from the base of the n\yel encephalon
to the wing buds was cut into 4 - 5 pieces and incubated in 20 ml
of the medium of Karnovsky and Roots for 1 hour at 38°C. After
incubation the tissue was dehydrated through a graded series of
ethanol, embedded in paraplast, cut at 10 urn and mounted on slides.
The reaction penetrated the tissue blocks slowly, so the reaction
product was only near the cut surface.
Tissue for the in vitro tests was from embryos incubated 3
days (average stage 19+). The tissue was incubated with 3 inhibitors
and 2 substrates (Table 3) to determine the specifity of the reaction.
Embryos for the in ovo tests were incubated for 3 days before
treatments. Three hours after treatment the embryos were removed
from the eggs and fixed as above. Some embryos were stained for
AChE without fixation. The reaction results of the latter were
recorded and slides were not made. The embryos in the in ovo tests were treated with eserine sulfate, isoOMPA, 284C51 or monocrotophos
(Table 4). Control embryos were injected with distilled water.
Embryos were treated at 3 days of incubation with eserine
(130 jjg), isoOMPA (100 jug), 284C51 (100 ug to 5 mg), or monocrotophos
(100 ug). Control embryos were injected with distilled water.
The embryos were incubated an additional 3 days, then fixed in FAA.
The embryos were stained in toto for cartilage using the method of Ojeda et al_. (1970).
Correction of monocrotophos defects by 2-PAM
Pyridine-2-aldoxime methiodide (2-PAM) was obtained from
Sigma Chemical Company.
White Leghorn embryos were treated at various times during
incubation with monocrotophos and 2-PAM. Embryos were pretreated
with 2-PAM on 2 (1 mg) or 3 (0.5 mg) days of incubation before an
injection of both monocrotophos (100 ug) and 2-PAM (1 mg) at 3 days
of incubation. Embryos were treated with both monocrotophos and
2-PAM at 3 days, and then 2-PAM (1 mg) at 4 days. Embryos were also
treated with monocrotophos alone (100 jug) or with an injection of
both monocrotophos and 2-PAM (1 or 2 mg) at 3 days of incubation.
Control embryos were injected with distilled water.
Embryos treated with both monocrotophos and 2-PAM (1 mg) were
removed 3 hours after treatment and fixed in 4% formalin plus 5mM
CaCl2 * The neck region was cut into 3 - 4 pieces and stained for
AChE using the method of Karnovsky and Roots (1964). The tissue was embedded and slides were made.
The remaining embryos were removed at 6 days of incubation and fixed with FAA overnight. Embryos were stained for cartilage in toto using the method of Ojeda et al_. (1970). The embryos were cleared in benzene and photographed. R esults
Gross effects of monocrotophos
Table 1 contains the measurements of treated and control embryos at 17 days of incubation. A t-test analysis for 2 means (Steel and
Torrie 1960) was conducted to compare populations. Data were reported as mean * standard deviation.
There was no significant difference (P>0.05) between treatments on 2 or 3 days of incubation. All the following comparisons are between treated and control embryos on the same treatment day. There was no significant difference (P>0.05) between treated and control embryos in head or beak lengths. The wings were significantly shorter in the treated embryos on 2 (P<0.02) and 3 days (P<0.0T) than in controls.
The toe length of the treated embryos was significantly shorter
(P<0.01) at 3 days and highly significant (P^O.OOl) at 2 days. The body, leg, and neck lengths were highly significant (P40.001) when compared to the controls at both 2 and 3 days of treatment.
Defect appearance versus day of treatment
The control embryos for treatments before incubation (Plate I,
Fig. 1) or at 1 day (Plate II, Fig. 4), 2 days (Plate III, Fig. 6),
3 days (Plate IV, Fig. 8}, 4. days (Plate V, Fig. 10), or 5 days
(Plate VII, Fig. 13) of incubation had the same appearance. The
19 Table 1: Comparison of body parameters for 2 and 3 day treatments (mm)
Day Body Leg Winq Neck Treated Control Treated Control Treated Control Treated Control
2 75.0±4.4# 86.0-0.9 47.8-3.3^ 53.5-1.3 27.3—1.3 * 28.4-0.5 9.8-1.9^ 20.7-1.5
3 77.3±2.7# 85.8-3.3 49.3-2.7^ 53.5-3.0 27.6-1.1* 28.8-1.3 11.8-2.0# 20.7-1.5
Day Head Beak Toe Treated Control Treated Control Treated Control
2 29.4-0.9 30.3-0.5 5.0-0.0 5.2-0.5 12.9-1.4^ 14.8-0.4
3 29.3-0.7 30.2-1.0 5.0-0.2 5.2-0.4 13.1-0.8* 14.2-1.0
P<0.02
* P<0.01
# P<0.001
Comparisons are between treated and controls of the same day 21
vertebrae were well developed with rectangular vertebral centra
and distinct neural arches. The notochord ran through the middle of
the vertebral centra. The neural tube was straight, with no infoldings
into its central canal.
Some of the embryos treated with monocrotophos before incubation
had the same appearance as controls of the same time period (Plate I,
Fig. 2). Others has a slightly warped notochord (Plate I, Fig. 3).
The centra of the vertebrae in the latter were slightly compressed.
Little or no fusion of the neural arches occurred when the embryos
were treated before incubation.
Treatment at 1 day of incubation increased the severity of the
defect at 6 days when compared to embryos treated before incubation.
There was increased warping of the notochord, and more vertebrae were
compressed (Plate II, Fig. 5). Fusion of both the neural arches
and vertebral centra occurred in the extreme points of the curvatures
of the notochord, involving 3 to 4 vertebrae.
Treatments at 2 days of incubation resulted in a more sinusoidal
notochord than observed previously (Plate III, Fig. 7b). Severe
compression of both the vertebrae and spinal ganglia was present
(Plate III, Fig. 7a). Fusion of the first 8 cervical vertebrae occurred both in the neural arches (Plate III, Fig. 7a) and in the
vertebral centra (Plate III, Fig. 7b).
Treatment of embryos at 3 days of incubation resulted in defects similar to those obtained with treatment at 2 days. There was fusion of the neural arches (PlatelV, Fig. 9a,b) and the vertebral centra
(Plate IV, Fig. 9a,b; Plate V, Fig. 9c). The sinusoidal path of the 22
notochord was mainly lateral. The neural tube showed evidence of twist
ing along with the notochordal curvatures.
Treatment of the embryos at 4 days of incubation resulted in
appearances ranging from a sinusoidal notochord and slight fusion
of the vertebral centra (Plate VI, Fig. 11) to a straight notochord
and no fusion of the centra (Plate VI, Fig. 12). Even with a
twisted notochord, the centra of these vertebrae were not compressed
as much as those of 3 day treatments. The neural arches in both
cases were fused from the second through the eleventh vertebrae. The
neural tube was twisted when the notochord was sinusoidal, similar to
that seen in embryos treated at 3 days. When the notochord was
straight, the neural tube was straight.
Five day treatments affected only the neural arches. The
neural arches of the first 10 to 12 cervical vertebrae were fused
(Plate VII, Fig. 14). The notochord and the vertebral centra
were straight as controls, not fused or compressed. The ganglia
were not compressed.
Development of defects with treatment at 3 days of incubation
Table 2 shows the average stage of the embryos and the per cent
of the embryos with defects as observed using a dissecting microscope
at the hour after treatment indicated. Embryos were staged after
membrane removal according to the system of Hamburger and Hamilton
(1951).
Three hours after treatment with monocrotophos was the earliest time period in which the embryos were observed. When the embryos were observed using the SEM, a variety of appearances were present. Table 2: Stage of development and per cent abnormal embryos at
various hours after treatment with monocrotophos
at 3 days of incubation9
Hour after Average^ % Abnormal treatment st'aqe Present Absent Questionable
3 20+ 39.1 56.5 4.3
6 21+ 83.3 11.1 5.5
9 22+ 84.2 15.8 0.0
12 22+ 71.4 21.4 7.1
24 24 100 0.0 0.0
36 26- 100 0.0 0.0
48 27 100 0.0 0.0
60 28 100 0.0 0.0
72 29 100 0.0 0.0 a Embryos were observed using a dissecting microscope b Stages according to Hamburger and Hamilton (1951)
+ or - indicates stages in between the numbered ones 24
The treated embryos ranged in appearance from the same as controls
(Plate VIII, Fig. 16) to compression of somites (Plate VIII, Fig. 18).
Embryos with slight compression were also observed (Plate VIII,
Fig. 17). Saggital sections of the treated embryos revealed a
slight compression of the area just external to the somites (Plate IX,
Fig. 20a). There was also a decrease of the area in between the
ganglia (Plate IX, Fig. 20b). The notochord did not deviate from
its normal position. A curve in both the neural tube and notochord
was indicated by the change in cut of the neural tube as it goes
from longitudinal to almost cross section to longitudinal again.
Some embryos were more affected than this at 3 hours after treatment.
A curve was still seen in the neural tube and notochord (Plate X,
Fig. 21), and the notochord had developed a sinusoidal appearance.
Control (Plate X, Fig. 22) and treated (Plate X, Fig. 23)
embryos were cut at 1 micrometer for light level observations.
The defect can be seen as a slightly misshapen notochord.
Cytoplasmic differences were observed at the ultrastructural level. Both the microtubules and microfilaments from the ventral
portion of the marginal layer of the neural tube were disturbed
(Plate XI, Fig. 25) in the monocrotophos treated embryos. The cytoplasm of the notochord in the control embryos (Plate XI, Fig. 26) was denser than that of the treated embryos (Plate XI, Fig. 27).
The treated embryos had fewer myofibrils (Plate XII, Fig. 29) than the controls (Plate XII, Fig. 28). The dark staining filaments are myosin and the fine ones around the myosin and scattered throughout the cytoplasm are actin (Allen 1978). The sclerotome around the 25 notochord of both the control (Plate XII, Fig. 30) and the treated embryos (Plate XII, Fig. 31) were similar. Extracellular fibril production was not as yet evident.
At 6 hours after treatment the compression of the somitic areas in the treated embryos (Plate XIII, Fig. 33) as observed using
SEM had increased over that observed at 3 hours after treatment. The treated embryos had side to side curvatures, as well as compression.
Light level slides demonstrated the same compression in the treated embryos (Plate XIV, Fig. 36a) as seen in SEM studies. The somites and ganglia were compressed in the treated embryos (Plate XIV,
Fig. 36b,c,e)when compared to the controls (Plate XIII, Fig. 34c; 35a,b).
The curvatures of the notochord (Plate XIV, Fig. 36d; 37) have increased in severity from that seen at 3 hours after treatment.
The neural tube was also twisted, as indicated by the warped appearance of the central canal.
The notochord of the treated embryo was off center and was pushing into the neural tube (Plate XV, Fig. 39) in the cross section.
The number and size of the vacuoles in the notochord of both treated and control embryos (Plate XV, Fig. 38) was similar.
The microtubules of the processes from the marginal layer of the neural tube appeared disturbed in the treated embryos (Plate XV,
Fig. 41) at the TEM level. The notochord cytoplasm was denser in the controls (Plate XVI, Fig. 42) than in the treated embryos (Plate XVI,
Fig. 43; 44). The notochordal curvatures were severe enough in places to obtain 2 sections of notochord in the same area (Plate XVI,
Fig. 44). The developing fibrils fn between the sections are collagen and glycosaminoglyeans (GAG), mainly chondrotin-4- and 6-sulfate 26
(Cohen and Hay 1971; Hay and Meier 1974). The developing myo- fibrils in the myotome of the treated embryos (Plate XVII, Fig. 45) were completely disorganized. Dark staining myosin filaments were present, but the arrangement was completely disrupted when compared to control embryos (Plate XVII, Fig. 45). Actin filaments were present in the cytoplasm of both treated and control embryos, but were more distinct in the controls. The sclerotome cells.of the control embryos (Plate XVII, Fig. 47) were oriented around the notochord. The sclerotome of the treated embryos from the same level (Plate XVII, Fig. 48) was not as well organized as the control.
The compression that was evident at the earlier time periods was still present at 9 hours after treatment (Plate XVIII, Fig. 50).
Internally the degree of curvature of the notochord had increased
(Plate XIX, Fig. 52a; 53) in the treated embryos. Usually the noto chord curved both laterally and dorsal-ventrally, but in some embryos a large portion of the notochord was in the same plane. In either case the notochord pushed into the neural tube forcing it into curves
(Plate XIX, Fig. 52a; 53b). The central canal of the neural tube was twisted. The ganglia were compressed with decreased area between them.
The myotome was compressed also.
In the cross sections the defect is seen as an off center notochord
(Plate XX, Fig. 55). There is a greater distance between the neural tube and notochord in the treated embryo than is found in the control
(plate XX, Fig. 54). The microfilaments of the neural tube were disorganized (Plate XX, Fig. 57) in the treated embryos at the EM level. The microtubules were thinner in some processes of the treated embryos than in the controls (Plate XX, Fig. 56). These processes were from the ventral portion of the marginal layer of the neural tube.
The cytoplasm of the cells in both the treated CPI ate XXI,
Fig. 59} and control CPI ate XXI, Fig.. 58) embryos had decreased in
density from that seen at earlier times. The control embryos had
slightly denser notochordal cytoplasm than the treated embryos. The
myotome of the treated embryo in this example (Plate XXI, Fig. 61)
was not as disrupted as the treated myotome of earlier times. The
myosin filaments of the treated embryos were less dense than those of
the controls (Plate XXI, Fig. 60). There were no observable differences
in the sclerotome of the treated (Plate XXII, Fig. 63) and control
(Plate XXII, Fig. 62) embryos. Both were oriented around the notochord.
The compression of the cervical region of the treated embryos
had continued to increase (Plate XXII, Fig. 65) by 12 hours after
treatment when compared to earlier times. The segments in the
treated embryos were much more distinct than in the controls (Plate XXII,
Fig. 64). These same segments could be seen in the saggital sections
(Plate XXIII, Fig. 67a). The myotome (Plate XXIII, Fig. 67b) and spinal ganglia (Plate XXIII, Fig. 67c) of the treated embryos were compressed, with a decrease in the area between the ganglia. The notochord and neural tube were both sinusoidal (Plate XXIII,
Fig. 67d, e; 68) with the curvatures of the notochord becoming more severe with compared to earlier times. The notochord curved laterally as well as dorsal - ventrally. It was pushing into the neural tube at the extremes of the curvatures. Infoldings occurred in the central canal of the neural tube and were most severe at this time period.
The cross sections of the treated embryos (Plate XXIV, Fig. 70) at this time display numerous defects. The notochord is oval, not 28 round as in the control (Plate XXIV, Fig. 69). The neural tube is twisted and the myotome is disrupted.
Again the processes of the marginal layer of the neural tube in the treated embryos as seen at the EM level are thinner and dis rupted (Plate XXIV, Fig. 72) when compared to the controls (Plate XXIV,
Fig. 71). Fine actin filaments were observed in the myotome of the treated embryos (Plate XXV, Fig. 74) but the myofibrils were completely disrupted. The cytoplasm of the notochord cells in the control embryos (Plate XXV, Fig. 75) is denser than that of the treated embryos (Plate XXV, Fig. 76). The sclerotome of both treated
(Plate XXVI, Fig. 77) and control (Plate XXVI, Fig. 78) embryos is similar.
The internal sinusoidal appearance of the cervical region was evident externally by 24 hours after treatment when the embryos were viewed using SEM (Plate XXVI, Fig. 80). The sections were becoming wedged shaped in order to fit into these curves. Internally the extreme lateral warping of the notochord was evident (Plate XXVII,
Fig. 82a,b). Pieces of the notochord were located beneath the ganglia, instead of being ventral to the neural tube as in the controls
(Plate XXVII, Fig. 81d). The neural tube was twisted and convoluted in the treated embryos, while the infoldings into the central canal had decreased. The ganglia of the treated embryos (Plate XXVII.
Fig. 82a; 83) were compressed with little or no space between them.
The myotome was also compressed. The areas which will develop into the vertebral centra were becoming defined. These areas in the treated embryos were compressed when compared to controls (Plate XXVII,
Fig. 81c,d). 29
The neural tube was elongated and the central canal slightly
twisted in the cross sections of the treated embryos (Plate XXVII,
Fig. 85). The notochord was oval, not round as in the controls
(Plate XXVIII, Fig. 84). There was a greater distance between neural
tube and notochord of the treated embryo in this section, than in the
control. Sclerotomic cells were oriented around the notochord and
were developing into chondroblasts in both the treated and control
embryos.
The results at the EM level were similar to those already dis
cussed for earlier times after treatment. Both the microtubules and
microfilaments found in the marginal layer of the neural tube
(plate XXVIII, Fig. 87) were disrupted in their organization. The
cytoplasm density of the notochordal cells of the controls (Plate XXIX,
Fig. 88) had decreased from earlier times, with increased amounts of
RER present. The notochordal cells of both the treated (Plate XXIX,
Fig. 89) and control embryos had the same cytoplasmic density. The myofibrils of the treated embryos (Plate XXX, Fig. 91) were starting to
recover from the effects of the pesticide treatment one day earlier, but these fibrils were still smaller and less numerous than those of the control embryos (Plate XXX, Fig. 90).
The cervical region of the treated embryos (Plate XXX, Fig. 93) as observed using the SEM was much shorter than the same area of the controls (Plate XXX, Fig. 92) by 1 \ days.after treatment with mono crotophos. The first 11 segments were extremely compressed. These same segments were hard to see in the controls. The internal appearance was very similar to that found 1 day after treatment. The notochord was extremely sinusoidal (Plate XXXI, Fig. 95), pushing into the neural tube as well as curving laterally. The ganglia and myotomic areas were compressed. The developing muscle was sparse in the treated embryos. Chondrification was continuing in both the treated and control embryos, with dense caudal and less dense cranial sclerotomic areas present in the controls (Plate XXXI, Fig. 94).
These same areas were compressed in the treated embryos, especially in the areas of maximum notochordal curvature.
Vacuolation of the notochords has increased with development, with no observable difference between the rate in the treated (Plate XXXI,
Fig. 97) and control (Plate XXXI, Fig. 96) embryos. Along with increased vacuolation, a decrease in cytoplasmic density of the noto chordal cells occurred. This was observable at the EM level in both treated (plate XXXII, Fig. 99) and control (Plate XXXII, Fig. 98) embryos. The myotome of the treated embryos (Plate XXXII, Fig. 101) had fewer myofibrils than found in the controls (Plate XXXII, Fig. 100), but seemed to be recovering from the effects of the pesticide.
Extreme shortening of the cervical region was observed in the treated embryos (Plate XXXIII, Fig. 103) by 2 days after treatment.
This region was one half the length of the same area in the control embryos (Plate XXXIII, Fig. 102). Internally chondrifi cation was proceeding normally around the notochord in both the control
(Plate XXXIII, Fig. 104) and treated (Plate XXXIII, Fig. 105b) embryos.
These areas were extremely compressed in the treated embryos. The notochordal curvatures were so extreme in places that a Z-shaped noto chord was seen. The neural tube was twisted at this same location.
The spinal ganglia were very close together. The ganglia touched in some cases. 31
Chondrification continued with the vertebral centra and neural arches (Plate XXXIV, Fig. 106) becoming distinct by Zh days after treatment (5^ days of incubation). The centra of the control embryos were rectangular in shape with lines of demarcation between them.
These same areas in the treated embryos were compressed (Plate XXXIV,
Fig. 107). There were indications of fusion of the vertebral centra in the treated embryos in the areas of extreme curvature of the noto chord. The ganglia were compressed and on top of each other in the same area.
Much fusion of the developing vertebrae was evident in the treated embryos by 3 days after treatment (Plate XXXIV, Fig. 109).
Fusion of the neural arches of the first 8 vertebrae was present.
Fusion of the vertebral centra was also evident. The centra have developed around the sinusoidal notochord and are themselves sinusoidal.
The centra were wedged-shaped in the extreme curves in order to fit " into the available area. The vertebrae of the treated embryos were much smaller than the controls (Plate XXXIV, Fig. 108). Muscle development in the treated embryos was affected, with less muscle present in the treated than in the control embryos. The ganglia were severely compressed and ran together in the areas of extreme notochordal curvatures.
The cartilagenous vertebrae were well developed by 5 days after treatment (8 days of incubation). There was a great difference in the lengths of the cervical regions of the control (Plate XXXV, Fig. 110) and treated (Plate XXXV, Fig. Ill) embryos. The neural arches were completely fused lateral and dorsal to the neural tube. The vertebral centra were fused through this region and shorter than t.he controls. 32
The extreme warping of the notochord was still evident. The developing muscle in the treated emhryos was less abundant than in the controls.
Histochemical stain for acetylcholinesterase
The results of the histochemical stain for AChE after in vitro treatment by inhibitors are contained in Table 3. Using acetyl thiocholine iodide (ACH) as a substrate, a strong positive stain was located in the developing myotome, in the marginal layer of the neural tube, in the ventral horn of the neural tube, and in the sympathetic chain ganglia (Plate XXXVI, Fig. 112). No substrate resulted in no stain (Plate XXXVI, Fig. 113), so nonspecific stain was not a problem.
Eserine treatment resulted in no stain reaction (Plate XXXVI, Fig. 114), indicating inhibition. IsoOMPA did not inhibit the formation of the reaction product (Plate XXXVI, Fig. 115). The positive areas were the same as in the controls. A range of 1,5-bis(4-ally1 dimethyl ammonium- phenyl )-pentane-3-l dibromide (284C51) from 110 u.g to 2 mg was tested.
The lower levels (110, 200 and 500 u^) were positive in a decreasing degree. Levels of 750 ua and higher amounts inhibited the reaction
(Plate XXXVII, Fig. 116).
A general background- stain resulted when butyryl thiocholine iodide (BuThCh) was used as a substrate (Plate XXXVII, Fig. 117).
Treatment with isoOMPA (Plate XXXVII, Fig. 118) and. 284C51. (PI ate XXXVII,
Fig. 119) resulted in the same appearance as the controls.
The results of the histochemical test for AChE after in ovo treatments at 3 days of incubation are located in Table 4. The control embryos with no inhibition had a strong.positive reaction (Plate XXXVII,
Fig. 120), with the same positive areas as were found in the in vitro. 33
Table 3: Histochemical test for acetylcholinesterase of tissue from
the cervical region of stage 19+ chicken embryos treated
in vitro with inhibitors
Inhibitor Amount Substrate Results Figure #
none ACH3 +++ 112
none none - 113
eserine 130 ucj (10“5M) ACH - 114
isoOMPA1 68 ug (10~5M) ACH +++ 115
284C512 .110 ug (10-5 m) ACH +++
284C51 200 ug ACH ++
284C51 500 ug ACH +
284C51 750 ug ACH - 116
284C51 l mg ACH -
284C51 2 mg ACH -
none BuThCh4 ± 117
isoOMPA 68 ug (10"5M) BuThCh ± 118
284C51 110 ug (10“5M) BuThCh ± 119
1 isoOMPA = tetraisopropylpyrophosphoramide
2 284C51 = 1,5-bi s(4-allyldimethylammoni umphenyl)-pentane-3-1 dibromide
3 ACH = acetyl thiocholine iodide
4 BuThCh = butyryl thiocholine iodide
+ to +++ = degrees of positive reaction
- = questionable reaction, but more positive than negative
- = negative reaction 34
Table 4: Histochemical tests for acetylcholinesterase after in ovo
treatments of stage 19 - 20 chicken embryos
Treatment Amount Fixative
None Formalin
Results Fiqure # none ++ ++ 120 eserine 130 ug - - isoOMPA1 100 ug + ++ isoOMPA 200 ug + ++ 121
284C512 300 ug ++ 122
284C51 1 mg * monocrotophos 100 ug - - 123
* isoOMPA = tetraisopropylpyrophosphoramide
^ 284C51 = 1,5-bis(4-all.yldimethylammoniumphenyl)-pentane-3-1 dibromide
Acetyl thiocholine iodide was used as the substrate in these tests.
+ to ++ = degrees of positive reaction f = questionable reaction, but more negative than positive
- = negative reaction 35 test. The stain was less intense with no fixative, but the areas stained were the same. Eserine inhibited the enzyme, so no reaction product was present. Treatment with isoOMPA did not inhibit the enzyme reaction (Plate XXXVII, Fig. 121). Dose levels higher than
200 utj/egg caused embryo mortality. The inhibitor 284C51 at 300 u.g/egg did not inhibit the enzyme reaction (Plate XXXVII, Fig. 122) when the tissue was fixed in formalin. The reaction was inhibited at 1 mg/egg in the first 30 minutes of incubation in unfixed tissue, but not after this time. Monocrotophos inhibited the enzymatic reaction
(Plate XXXVIII, Fig. 123).
The embryos that were stained for cartilage at 6 days of incubation after treatment at 3 days had the following appearances. Control embryos had straight vertebrae with no fusion (Plate XXXVIII, Fig. 129;
Plate XXXIX, Fig. 130). Monocrotophos treated embryos had the expected appearance (Plate XXXVIII, Fig. 124) of a sinusoidal and compressed cervical region with fusion. Embryos treated with isoOMPA
(Plate XXXVIII, Fig. 125) were normal. Embryos treated with 284C51
(Plate XXXVIII, Fig. 127; 128) had the same appearance as the controls throughout the treatment range (100, 200, 300 ucj, 1, 2.5, 5 mg/egg).
Correction of monocrotophos defects by 2-PAM
Control embryos had straight vertebral columns, with no curvatures or fusion (Plate XXXVIII, Fig. 129; Plate XXXIX, Fig. 130). Mono crotophos treated embryos exhibited the characteristic syndrone
(Plate XXXIX, Fig. 131; 132) with fused and compressed vertebrae, a shortened cervical region and extremely sinusoidal appearance of the axial skeleton in the cervical region. The fusion involved the dorsal 36
portion of the neural arches of the second to sixth or seventh
vertebrae.
Embryos treated with both monocrotophos and 2-PAM Cl or 2 mg/egg)
at 3 days of incubation exhibited a range of appearances. These
embryos varied in appearance from being similar to the controls
(Plate XXXIX, Fig. 133) to having fused and sinusoidal vertebrae
(Plate XXXIX, Fig. 135) as seen in the monocrotophos treated embryos.
Most of the embryos treated in the above manner displayed an appearance
intermediate to the control and monocrotophos treated embryos. The
curvature of the cervical region was present. Fusion, if present,
occurred between 2 or at most 3 vertebrae. The intervertebral spaces
were larger than in the treated embryos, but still smaller than in the
controls. The cervical region was increased in length with 2-PAM
treatment.
Embryos pretreated with 2-PAM on either 2 or 3 days of incubation
before treatment with both monocrotophos and 2-PAM on 3 days of incu
bation still had the cervical curvatures. Fusion occurred very rarely
or was absent in these embryos (Plate XL, Fig. 136). Others treated
in this manner appeared normal (Plate XL, Fig. 137).
Embryos treated with monocrotophos and 2-PAM on 3 days of incu
bation and with 1 mg of 2-PAM 24 hours later (Plate XL, Fig. 138; 139) '
appeared similar to the above embryos. The warping of the cervical
region decreased from that seen in monocrotophos treated embryos, and
fusion, if present, occurred between 2 or 3 vertebrae.
The histochemical stain for AChE was negative, with no reaction
product formed (Plate XL, Fig. 140) when the embryos were treated with both 2-PAM and monocrotophos. Discussion
Gross effects of monocrotophos
Treatment with monocrotophos caused a shortened body length, mainly resulting from a shortened neck. The necks of the treated embryos were half the length of the necks of the control embryos. The body length minus the neck length was the same in the controls and treated embryos. Shortening of the extremities occurred, with the legs affected more than the wings. The legs were not defective in any way other than length. No head or beak defects were noted.
Monocrotophos has been reported to cause parrot beak, shortened and deformed legs, growth retardation, and shortened neck (Roger
1967; Roger et aK 1969). The full range of defects depended upon the dose level, with increasing severity of the defects with increasing dose (Roger et al_. 1969). No head or beak defects were found in this study at the dose level used.
Defect appearance versus day of treatment
The results obtained in this experiment were different from those obtained by Roger et al_. (1964; 1969). The time of treatment did influence the resulting defect. The amount of pesticide used was probably the key, because the degree of defect is dose related. The effects due to treatment at a specific time were masked with an increasing pesticide dose. 37 38
The time of treatment had a distinct influence on the appearance
of the defect at 6 days of incubation. Treatment before incubation
resulted in no defects, or a slight defect in the notochord and
vertebral centra. The neural arches were slightly affected, if at all.
Roger et al_. (1969) stated that dosages of 0.3 mg or higher of monocro
tophos have to be given prior to incubation to cause teratogenic
effects.
Treatment at 1 day of incubation increased the severity of the
defects. Compression of the vertebral centra, and warping of the
notochord resulted. Increased fusion of the neural arches was present.
Two and 3 day treatments resulted in similar defects, which were more
severe than those seen in earlier treatments. There was fusion and
compression of the vertebrae, compression of the spinal ganglia, and
an extremely sinusoidal appearance of the notochord. Treatment at 4 days
resulted in a lessening of the defects. The defects shifted from the ventral to the dorsal portion of the developing vertebrae. The centra were not as compressed as earlier and notochord curvatures had lessened. Fusion of the neural arches was present while fusion of the vertebral centra had lessened. Treatments at 5 days affected only the neural arches. No shortening of the cervical region or fusion of the vertebral centra was present with treatments at 5 days.
The insecticide affected the ventral portion of the vertebrae when the embryos were treated at 1, 2, or 3 days of incubation.
The neural arches were still affected at 4 days of incubation, but with a lessening of the effects in the vertebral centra. The defects shifted to the dorsal portion of the vertebrae with 5 day treatments.
The most sensitive treatment times were around 3 days of incubation. 39
Roger et al_. (1964) reported that treatment of chicken embryos
with dicrotophos during the first 6 days of incubation resulted in the
same cervical defects. These embryos were observed (externally only)
at 21 days of incubation. Roger et al_. (1969) treated chicken embryos
on 0, 2, or 4 days of incubation with 0.3 mg of monocrotophos. No
differences were found in the resulting abnormalities. Four days of
incubation was found to be the most sensitive to both dicrotophos
and monocrotophos treatments. Treatment after 6 days of incubation
resulted in no defects.
Development of defects with treatment at 3 days of incubation
The first indications of the cervical defect were seen by 3 hours after treatment. The somitic areas of this region were compressed or reduced in size, causing an infolding of the epidermis to outline the areas. Neither the notochord nor the neural tube was strongly < s affected. As development continued this exterior compression remained and increased in severity.
Internally the myotome areas of the somites became compressed shortly after treatment. The myofibrils of the developing muscle were disrupted in the treated embryos within 3 hours of treatment. They recovered later in development, but the resulting muscle was less abundant than in the controls.
The spaces between the cervical ganglia were smaller in the treated than in the control embryos, with the ganglia themselves becoming compressed with further development. This compression became so severe that some ganglia ran together before joining the neural tube. The f irs t indication of notochordal involvement was a curve near
the level of the eighth cervical vertebrae. This curve became sharper
with further development with warping occurring in a lateral as well
as dorsal - ventral direction. This warping increased in severity
until chondrification occured. Notochord vacuolation occurred in
both treated and control embryos at the same pace. The cytoplasm of
the treated notochord cells was less dense than the controls shortly
after treatment, but the cytoplasm of both became less dense as the
vacuolation continued. This difference in density was in the ground
substance of the cytoplasm.
The neural tube was also affected by insecticide treatment. The
early notochordal curve was paralled by a similar curve of the neural
tube. The notochord appeared to push into the neural tube at the apex of its curves. There was a twisting and infolding in the central
canal of the neural tube. These were most severe around 12 hours after
treatment, then after this time the infoldings decreased. The
pesticide seemed to have a direct effect on the marginal layer of the neural tube. Both the microtubules and microfilaments of this area were disrupted.
Early chondrification sequences occurred throughout the time of
this study. Cartilage that was recognizable as such at the light level started to develop around 4 to 5 days of incubation. Chondrification timing did not appear to be influenced by monocrotophos treatment. The vertebrae developed around the notochord, using i t as a template. The vertebrae of the treated embryos were compressed and wedge - shaped as
they developed. They were forced into the wedges in order to follow the 41
curvatures of the notochord. Fusion occurred between these compressed
vertebrae. The developing neural arches were also fused. A solid
ring of cartilage had developed around the neural tube by 5 days after
treatment. This extended from the first to the eighth or tenth
cervical vertebrae. This cartilagenous mass was thicker than normal
vertebrae and about half the length. The mass of fused vertebral
centra were no longer warped, because cartilage had filled in between
the curves. The notochordal curves were s till present.
Schom and Abott (1977) reported an abnormal bending in chicken
embryos treated with monocrotophos at stage 20. This bending was in
the same region as the first signs of compression noted in the
present study.
Nicotine sulfate treatment of chicken embryos at 2 or 4 days of
incubation (Strudel 1971; Strudel and Gateau 1971) caused notochordal defects similar to those reported in the present work. The neural tube and somites were not affected until 24 hours after treatment. The defects at 48 hours after treatment were very similar to the ones obtained in this work at that time period. The authors concluded that the notochord was responsible for the defect.
Meineil (1970; 1973a; 1973b; 1974a; 1975b; 1977a) treated chicken eggs with parathion at 0, 1, or 2 days of incubation. A sinusoidal notochord at 4 days of incubation was the first defect noted. The neural tube was also affected by this time. The somites were irregular by 5 days. The defects after 6 days of incubation were similar to those obtained in the present experiment.
These authors reported notochordal defects as the firs t teratogenic signs after treatment with insecticides. In the present study the firs t 42
signs were a compression or shortening of the somitic areas and the
region just lateral to the somites with an increased outlining of the
same externally. The bending observed by Schom and Abott (1977) would
correlate with these results.
The compression of the somitic areas might have caused less than
normal space for the notochord to elongate. The notochord development was not hindered by the insecticide, so i t continued to elongate. The
notochord bent in order to f it into the smaller than normal space. This was the first bending of the notochord. The notochord continued to grow in length in a smaller than normal space, so increased warping occurred. A 3-dimensional effect would be expected, and the evidence verified this.
The notochord pushed into the neural tube causing some of the twisting seen in it. The neural tube might also be directly affected by the insecticide thus impeding its normal development. Evidence for the direct effect would be the disorganized appearance of the microfilaments and microtubules after insecticide treatment. This could be another factor in the twisting of this structure.
The ganglia were compressed because there was less space between them than normal. They were unable to enlarge properly due to the limited space, so compression resulted.
The vertebral centra formed around the notochord. To f i t into the curves of the notochord, the centra developed as wedges. This disruption of the normal rectangular shape as well as the extreme compression might have caused fusion of the vertebral centra. Another factor to consider in the fusion of the vertebrae was lack of movement. 43
Drachman and Sokoloff C1966), Murry and Drachman (1969), and Sullivan
(1966) showed that lack, of movement during development caused joint fusion. The joint cavities failed to develop, and solid tissue resulted. This probably was instrumental in the fusion of the neural arches. Strudel (1955a; 1955b; 1967) reported that removal of the notochord caused a solid ventral mass of cartilage to form. The misshapen notochord created in this study might not have had the proper influence on the vertebrae and a solid mass formed.
The developing muscle was directly affected by the insecticide.
The myofibrils were disorganized shortly after treatment. The muscle recovered somewhat, but there were still fewer myofibrils than in the controls. Meiniel (1975b) reported the same effect using parathion.
He reported fewer myofibrils in treated embryos at 8 days of incubation
(treatment at 0 days) than in controls.
Histochemical stain for acetylcholinesterase
In vitro treatment with eserine prevented the formation of the reaction product. This finding indicates the general enzyme class cholinesterase (ChE) (Pearse 1972). IsoOMPA at 10"^M did not inhibit the enzyme when ACH was the substrate, while 284C51 did inhibit i t at 7 x 10"^M and higher levels. No reaction product was present when BuThCh was used as a substrate. These results indicate that true acetylcholinesterase was the enzyme responsible for the formation of the reaction product.
Drews (1975) reported the effective range for inhibition by
284C51 to be 1 x 10-5M to 2 x 10“4M. Pearse (1972) reported 100% inhibition of AChE in rat brain homogenate at 3 x 10“5 m 284C51 and 44
only 2% ChE inhibition at this, level. He also reported 90% ChE
inhibition with isoOMPA at 1 x 1Q“^M and 0% AChE inhibition.
The sites which were positive in the control embryos in this study were the same as reported by Drews (1975) for stage 20 chicken embryos.
These sites were nerve or muscle locations and would be expected to
contain AChE. The notochord did not stain for AChE using this
technique.
Both monocrotophos and eserine treatment in ovo at 3 days of
incubation completely inhibited the enzyme. The cervical region was
defective with treatment by these compounds at 6 days of incubation.
IsoOMPA did not inhibit the enzyme and no defects were observed in
the embryos. In ovo treatments with 284C51 inhibited the enzyme only slightly by 3 hours later. No defects were observed at 6 days of incubation using this compound, even at high dose levels. This compound is a reversible inhibitor of AChE (Drews 1975), while both monocrotophos and eserine are irreversible inhibitors. The slight inhibition was obtained by processing fresh tissue as rapidly as possible. Even then the reaction product formed after one half hour in the incubation media.
This study showed a correlation between inhibition of AChE or lack of it at 3 hours after treatment and resulting defects at 3 days after treatment. Meiniel (1975b) reported a similar correlation between AChE inhibition and resulting defects using malathion, dicrotophos, and parathion as inhibitors.
Acetylcholinesterase has been reported to be involved with functions other than nerve impulse transmission. Some authors suggested that i t was involved in cell movements (Drews 1975; Bishop 45 et^aK 1976). Others found a role for AChE in protein synthesis
(Satler ert aK 1974; Black and Reis 1971) or i.n the regulation of transport of substances CSastry et 1976). AChE activity was involved in development of both cardiac (Taylor 1976) and skeletal muscle (Filogamo and Gabella 1967; Drews 1975; Sawyer et a l. 1976).
Some of these functions might explain the presence of AChE in embryos before nerve activity occurres.
Correction of monocrotophos defects by 2-PAM
Wilson and Ginsburg (1955) reported pyridine-2-aldoxime (2-PAM) to be a powerful reactivator of acetylcholinesterase inhibited by organophosphates.
Counter treatment of monocrotophos treated chicken embryos with
2-PAM resulted in a range of defects. These range from fused vertebrae with extreme curvatures to straight vertebrae with no fusion as expected in controls. Increased levels of 2-PAM seemed to decrease the severity of the defects. Fusion was decreased or eliminated by using 2-PAM, but the curvature involving the vertebral centra remained in most cases.
Embryos treated with 2-PAM along with monocrotophos have longer cervical regions than embryos treated with monocrotophos alone.
Overall, 2-PAM treatment decreased the severity of the defects caused by monocrotophos, but did not eliminate them. AChE was not reactivated after a 3 hour treatment with 2-PAM in this study. This would correlate with the presence of the defect after 2-PAM treatments.
Pralidoxime, P2S, 2-PAM and TMB-4 reduced or eliminated the defects caused by treatment of quail eggs with parathion (Meiniel 1975b;
1976a; 1976b). These eggs were treated before incubation, not later in development when the embryos were more sensitive to the insecticides.
Pralidoxime countered the effects of parathion injected into chicken eggs on 5 days of incubation (Meiniel 1975b), but this was also not during the time the embryos were most sensitive to insecticide treatment. Conclusion
The first indication of a defect noted in this study was a
shortening of the somitic areas of the cervical region of monocro
tophos treated embryos, especially of the myotome and the region
lateral to the myotome. The muscle of this area was directly
affected by the insecticide as observed by ultrastructural and
enzyme studies. The arrangement of the actin and myosin filaments
of the myofibrils in the treated embryos was disorganized by 3 hours
after treatment at stage 20. No myofibrils were observed by 6 hours
after treatment. Acetylcholinesterase (AChE) is present in the
normally developing skeletal muscle at this time (Drews 1975) and
is involved in normal development of the muscle (Filogamo and
Gabel!a 1967). This enzyme is inhibited by the insecticide as
demonstrated by the histochemical stain.
Schom and Abott (1977) reported that treatment with monocro
tophos at stage 20 caused an abnormal bending of the cervical region
within 1 hour after treatment. This corresponds to the shortening
seen in the present experiments.
The embryos are most sensitive to insecticide treatment at
stage 20. This could be a very susceptible time in muscle development, when AChE is necessary for proper development. Normal embryo movements usually start around stage 22 to 24 (3% to 4 days of
incubation). 47 The decrease in length of the njyotome and the region lateral to it could have caused a decrease in the area available for notochordal elongation. Vacuolation and elongation of the notochord continued normally, and thus the notochord was forced into curves to fit into a less than normal space. When chondrification occurred, the developing vertebrae were wedge-shaped to fit around these curves.
Fused and compressed cervical vertebrae resulted. This area was about one-half the normal length, accounting for the reduction in neck and body lengths that were recorded in the 17 day embryos.
Lack of movement has been suggested as a cause of joint fusion
(Murry and Drachman 1969). Inhibition of AChE by monocrotophos perhaps causes this lack of movement, and therefore may contribute to the fusion of the vertebrae. The half-life of the insecticide in the eggs is around 2 days (Schom and Abott 1977), therefore the insec ticide may still be present later in development and continue to inhibit AChE.
The notochord appeared to push into the neural tube at the extremes of its curvatures. This might have contributed to the twisting of the neural tube. This twisting was most severe at 12 hours after treatment, then decreased in severity after this time.
The insecticide also appeared to directly affect the neural tube.
Evidence for this is the disruption of the microfilaments and micro tubules of the marginal layer of the ventral edge of the neural tube.
AChE is located in this area of the neural tube and is inhibited by the insecticide.
Treatments with pyridine-2-aldoxime methiodide (2-PAM) partially corrected the cervical defects caused by monocrotophos. The cervical region was longer with 2-PAM treatments, and in some cases fusion
did not occur. AChE is reactivated by 2-PAM because it forms a complex with the dimethyl phosphate portion of monocrotophos and thus frees the AChE. Dimethyl phosphate is the half of the insec ticide which attaches to and inhibits AChE.
Inhibition of AChE may have caused the observed defects due
to its role in skeletal muscle development. Two possible modes of action are indicated. If the muscle is functional at stage 20, then inhibition of AChE would cause a sustained contraction of the muscle
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Plate I
Fig. 1-3 Embryos injected before incubation then fixed after 6
days of incubation, a, neural arch; g, spinal ganglia;
m, muscle; n, notochord; t, neural tube; v, vertebral centra
10 urn sections.
Fig. 1 Control embryo 130X.
Fig. 2-3 Embryos treated with 100 tig monocrotophos.
Fig. 2 This embryo has the same appearance as the controls. 130X.
Fig. 3 Fusion of 2 neural arches occurred (large arrow). The
notochord has a slightly sinusoidal appearance (small
arrow). 130X. 57 58
Plate II
Fig. 4-5 Embryos treated at 1 day of incubation and fixed at 6 days
of incubation, a, neural arch; g, apinal ganglia;
m, muscle; n notochord; t, neural tube; v, vertebral
centra. 10wn sections.
Fig. 4 Control embryo 130X.
Fig. 5 Treated embryo injected with 100 ug monocrotophos. There
is an indication of neural arch fusion, and a warped
notochord is present. 130X. 59 60
Plate III
Fig. 6-7 Embryos treated at 2 days of incubation and fixed at 6
days, a, neural arch; g, spinal agnglia; m, muscle;
n, notochord; t, neural tube; v, vertebral centra.
lOum sections.
Fig. 6 Control embryos. Representative saggital sections. 130X.
Fig. 7 Treated embryos injected with 100 ucj monocrotophos. The
neural arches are fused and the ganglia compressed (arrow).
The space between the ganglia is less than in the control.
A sinusoidal notochord is evident. Representative
saggital sections. 130X. 61 62
Plate IV
Fig. 8-9 Embryos treated at 3 days of incubation and fixed at 6
days, a, neural arch; g, spinal ganglia; n, notochord;
t, neural tube; v, vertebral centra. lOum representative
saggital sections.
Fig. 8 Control embryos. 130X.
Fig. 9 Treated embryo injected with 100 ug of monocrotophos.
The large arrow indicates the same area in the sections.
The neural arches are fused and ganglia are compressed.
The neural tube and vertebral centra are sinusoidal. 130X 63
WJ in. 64
Plate V
Fig 9c This embryo was treated with 100 ug of monocrotophos
at 3 days of incubation then fixed at 6 days. Compressed
spinal ganglia (g) are present. The neural tube (t),
notochord (n)» and vertebral centra (v) are extremely
sinusoidal. The large arrow is indicating the same region
as found in Fig. 9a and 9b. 10 urn sections. 130X.
Fig. 10 Control embryos injected at 4 days of incubation and fixed
at 6 days, a, neural arch; g, ganglia; n, notochord;
t, neural tube; v, vertebral centra. 10 urn representative
saggital sections. 130X. 65 66
Plate VI
Fig. 11- Embryos treated at 4 days of incubation with 100 ug of
monocrotophos and fixed at 6 days of incubation. These
represent the extremes in appearance of the defect with
treatment at 4 days of incubation, a, neural arch;
n, notochord; t, neural tuber v, vertebral centra.
lOum sections.
Fig. 11 The neural arches are fused (large arrow). The neural
tube, notochord and vertebral centra (small arrow) are
extremely twisted. The vertebral centra are not as
compressed as those of earlier treatments. 130X.
Fig. 12 Fused neural arches (large arrow) are present as the only
defect. 130X. 67 68
Plate VII
Fig. 13- Embryos were treated a t 5 days of incubation and removed
at 6 days, a, neural arch; g, spinal ganglia; n, notochord;
t, neural tube; v, vertebral centra. 10 urn sections.
Fig. 13 Control embryo. 130X.
Fig. 14 Treated embryo injected with 100 ug monocrotophos. Fusion
of neural arches (arrow) is seen in these sections. The
remaining areas appear normal. Representative saggital
sections. 130X.
70
Plate VIII
Fig. 15-18 Embryos injected at 3 days of incubation and fixed 3 hours
a fte r treatment. A, anterior end of embryo. Size bar =
500 urn.
Fig. 15 Control embryo
Fig. 16-18 Treated embryos Injected with 100 ug of monocrotophos. 71 72
Plate IX
Ftg. 19- 20 Embryos were injected at 3 days of incubation and
fixed 3 hours later, g, spinal ganglia; m, myotome;
n, notochord; t, neural tube.
10 urn representative saggital sections.
Fig. 19 Control embryo 130X.
Fig. 20 Embryo treated with 100 ug monocrotophos. Compression
of the region external to the myotome (20a, arrows) is
present. 130X. 73 74
Plate X
Fig. 21- 23 Embryos injected at 3 days of incubation and fixed
3 hours later, g, spinal ganglia; m, myotome;
n, notochord; s, sclerotome; t, neural tube.
Fig. 21 Embryo treated with 100 ug monocrotophos. Curvature of
both notochord and neural tube is present. The arrow
is indicating the same position in the sections.
10 urn representative saggital sections. 130X.
Fig. 22 Control embryo, 1 urn cross section, 580X.
Fig. 23 Embryo treated with 100 ug monocrotophos, 1 urn cross
section. 580X. 75 Plate XI
Fig. 24- Marginal layer of the ventral edge of the neural tube.
Embryos injected at 3 days of incubation and fixed 3
hours later, f, microfilament; m, mitochondria;
t, microtubules.
Fig. 24 Control embryo. 6300X.
Fig. 25 Embryo treated with 100 ug monocrotophos. 6300X.
Fig. 26- Notochord. Embryos injected at 3 days of incubation
and fixed 3 hours later, er, endoplasmic reticulum;
m, mitochondria; n, nucleus; vac, vacuoles.
Fig. 26 Control embryo. 4700X.
Fig. 27 Treated embryo injected with 100 ug monocrotophos.
4700X. 77 78
Plate XII
Fig. 28- 29 Myotome. Embryos injected at 3 days of incubation and
fixed 3 hours later, my, myofibril.
Fig. 28 Control embryo. 6900X.
Fig. 29 Treated embryo injected with 100 ug monocrotophos.
6900X.
Fig. 30- 31 Sclerotome, oriented around notochord, er, endoplasmic
reticulum; n, nucleus.
Fig. 30 Control embryo. 7300X.
Fig. 31 Treated embryo injected with 100 ug monocrotophos.
7300X. 79
• v - : < ^
tp. ;. » T> wifc' •’/• >■ •V ' 4 % * %,/C ?=»■*:•. *5* % *;/*-
< ■**<.’i :l < :'.« ... '-At' *r \ < 1 * *. . * ’«*" V% tv;, i ’••■ *• • f/S r •( ’■pt*? ,< * *• ■.'’.-A jV v *v:-> • &&&•&:
■f’-w • - -.ri- - 80
Plate XIII
Fig. 32- 33 Embryos injected at 3 days of incubation and fixed 6
hours la te r. A, anterior end of embryo, size
bar = 500 urn.
Fig. 32 Control embryo.
Fig. 33 Treated embryo injected with 100 ug monocrotophos.
Compression of the somites is easily seen.
Fig. 34- 35 Control embryos, g, spinal ganglia; m, myotome;
n, notochord; t, neural tube. 10 urn representative
saggital sections. 130X. 81 82
Plate XIV
Fig. 36- Embryos treated at 3 days of incubation with 100 ug
monocrotophos and then fixed 6 hours la te r, g, spinal
ganglia; m, myotome; n, notochord; t, neural tube.
10 urn representative saggital sections.
Fig. 36 The arrow indicates the same location in the sections.
Compression of somites and ganglia is present along
with notochordal curvatures. 130X.
Fig. 37 Notochordal curvatures (arrow) are more severe than at
3 hours after incubation. 130X. 83 84
Plate XV
Fig. 38- Embryos injected at 3 days of incubation and fixed
6 hours later, m, myotome; n, notochord; s, sclerotome;
t, neural tube. 1 urn cross sections.
Fig. 38 Control embryo. 130X.
Fig. 39 Embryo treated with 100 ug monocrotophos. 130X.
Fig. 40- Marginal layer of the ventral edge of the neural tube.
Embryos injected at 3 days of incubation and fixed 6
hours later, m, mitochondria; t, microtubule.
Fig. 40 Control embryo. 6000X.
Fig. 41 Embryo treated with 100 ug monocrotophos. 6000X. 39 38 »' 1 , '• # " • t ' ,-y ’’A ' - x t / ^ * 44^ '
. ■/\.a „ * ;£ + -■ £ >■> * m •: A » 'rv i? '* ,A ,4i •t.Ai'l' >**V fJ '**#*
W lR .
. .- IM -«-^.»-f .•l i d * a/V m^ x ‘^1 y *%!jC * , v
...... a f e ^ ^ . 4 V .hvt -C/*.sK*«A^V. •;**,* " •.. • " .- ‘ :•/*•> ••• ,-y -
'•■T’ i v A" 7 ,:. V > „ *4 ’
• ■ * ^'A *, \ .Vi
•'v ■r\’CiS 86
Plate XVI
Fig. 42- Notochord of embryos injected a t 3 days of incubation
and fixed 6 hours later, gag, glycosami noglycans;
n, nucleus; vac, vacuole.
Fig. 42 Control embryo. 4000X.
Fig’. 43 Embryo treated with 100 ug of monocrotophos. 4000X.
Fig. 44 Embryo treated with 100 ug of monocrotophos. Two
sections of notochord are present, demonstrating the
severity of the curvature of the notochord. 3700X. 87
mm m m
$ $ i m 0 m m 88
Plate XVII
Fig. 45- ftyotome of embryos injected a t 3 days of incubation
and fixed 6 hours later, a, actin filaments;
my, myofibril.
Fig. 45 Control embryo. 4600X.
Fig. 46 Embryo treated with 100 ug monocrotophos. 4600X.
Fig. 47- Sclerotome oriented around the notochord, injected at
3 days of incubation and fixed 6 hours later, n, nucleus.
Fig. 47 Control embryo. 5700X.
Fig. 48 Embryo treated with 100 ug monocrotophos. 5700X.
90
Plate XVIII
Fig. 49- 50 Embryos injected at 3 days of incubation and fixed
9 hours la te r. A, anterior of embryo; size
bar = 500 urn.
Fig. 49 Control embryo.
Fig. 50 Embryo treated with 100 ug of monocrotophos.
Fig. 51 Control embryo injected at 3 days of fncubation then fixed
9 hours later, g, spinal ganglia; m, muscle; n, notochord;
t, neural tube. 10 urn representative saggital sections.
130X. 91 92
Plate XIX
Fig. 52- Embryos injected with 100 ug monocrotophos a t 3 days of
incubation, then fixed 9 hours later, g, spinal
ganglia; m, muscle; n, notochord; t, neural tube.
10 wn representative saggital sections.
Fig. 52 The notochord curvature has increased from that seen at
6 hours a fte r treatment. Compression of both the
ganglia and developing muscle is present. 130X.
Fig. 53 The notochord curves laterally (arrow) in addition to
dorsal - ventrally. 130X. 93 Plate XX
Fig. 54- Embryos injected at 3 days of incubation and then
fixed 9 hours later, g, spinal ganglia; m, myotome;
n, notochord; s, sclerotome; t, neural tube. 1 urn
cross sections.
Fig. 54 Control embryo. 580X.
Fig. 55 Embryo treated with 100 ug monocrotophos. 580X.
Fig. 56- Marginal layer of ventral edge of neural tube. Embryos
injected at 3 days of incubation and fixed 9 hours
later, f, microfilaments; t, microtubules.
Fig. 56 Control embryo. 8800X.
Fig. 57 Embryo treated with 100 £g monocrotophos. 8500X. 95
>■*
MI Plate XXI
Fig. 58-59 Notochord. Embryos injected at 3 days of incubation
and then fixed 9 hours later, n, nucleus.
Fig. 58 Control embryo. 6000X.
Fig. 59 Embryo treated with 100 ug monocrotophos. 7500X.
Fig. 60-61 Myotome. Embryos injected a t 3 days of incubation
and then fixed 9 hours later, tny, myofibrils.
Fig. 60 Control embryo. 6900X.
Fig. 61 Embryo treated with 100 ug monocrotophos. 6900X.
Plate XXII
Fig. 62-i Sclerotome that is oriented around the notochord. Embryos injected a t 3 days of incubation and then
fixed 9 hours later, er, endoplasmic reticulum;
n, nucleus.
Ftg. 62 Control embryos. 6600X.
Fig. 63 Embryo treated with 100 ug monocrotophos. 6600X.
Fig. 64- Embryos injected at 3 days of incubation and then
fixed 12 hours later. A, anterior end of embryo;
size bar = 500 um.
Fig. 64 Control embryo.
Fig. 65 Embryo treated with 100 ug monocrotophos. 99 100
Plate XXIII
Fig. 66- 68 Embryos injected at 3 days of incubation and then
fixed 12 hours later, g, spinal ganglia; m, myotome;
n, notochord; t, neural tube. 10 urn representative
saggital sections.
Fig. 66 Control embryo. 130X;
Fig. 67 Embryo treated with 100 ug monocrotophos. The myotome
and ganglia are both compressed. The neural tube and
notochord are sinusoidal. The neural tube has infoldings
into its central canal (arrow). 130X.
Fig. 68 An extremely curved notochord is present in this embryo.
130X. 101 Plate XXIV
Fig. 69- Embryos injected at 3 days of incubation and fixed
12 hours later, g, spinal ganglia; m, myotome;
n, notochord; t, neural tube. 1 urn cross sections.
Fig. 69 Control embryo. 580X.
Fig. 70 Embryo treated with 100 ug monocrotophos. The myotome
is disrupted and both the neural tube and notochord are
misshapen. 580X.
Fig. 71- Marginal layer of the ventral edge of the neural tube.
Embryos injected a t 3 days of incubation and fixed 12
hours later, t, microtubules.
Fig. 71 Control embryo. 7600X.
Fig. 72 Embryo treated with 100 ug monocrotophos. 7600X.
Plate XXV
Fig. 73- Myotome. Embryos injected a t 3 days of incubation
and fixed 12 hours later, a, actin filaments;
rny, myofibril.
Fig. 73 Control embryo. 6700X.
Fig. 74 Embryo treated with 100 ug monocrotophos. 6700X.
Fig. 75- Notochord. Embryos injected at 3 days of incubation
and fixed 12 hours later, n, nucleus; vac, vacuole.
Fig. 75 Control embryo. 4300X.
Fig. 76 Embryo treated with 100 ug monocrotophos. 4300X. 105 Plate XXVI
Fig. 77- Sclerotome. Embryos injected at 3 days of incubation
and fixed 12 hours later, n, nucleus.
Fig. 77 Embryo treated with 100 ug monocrotophos. 5100X.
Fig. 78 Control embryo. 5100X.
Fig. 79- Embryos injected at 3 days of incubation and fixed
1 day la te r. A, anterior end of embryo, size
bar = 500 urn.
Fig. 79 Control embryo.
Fig. 80 Embryo treated with 100 ug monocrotophos. 107 108
Plate XXVII
Ftg. 81—i Embryo injected a t 3 days of incubation and fixed
1 day later, g, spinal ganglia; m, myotome; n, notochord;
t, neural tube. 10 urn representative saggital sections.
Fig. 81 Control embryo. 110X
Fig. 82 Embryo treated with 100 ug monocrotophos. Warped neural
tube and notochord are present. 87X.
Fig. 83 Embryo treated with 100 ug monocrotophos. Compressed
ganglia and myotome occur in this section. 87X. 109 Plate XXVIII
Fig. 84- Embryos injected at 3 days of incubation and fixed
1 day later, g, spinal ganglia; m, myotome;
n, notochord; s, sclerotome; t, neural tube. 1 urn
cross sections.
Fig. 84 Control embryo. 580X.
Fig. 85 Embryo treated with 100 ug monocrotophos. Misshapen
notochord and neural tube are present. 580X.
Fig. 86 -1 Marginal layer of ventral portion of neural tube.
Embryos injected at 3 days of incubation and fixed 1
day later, f, microfilaments; t, microtubules.
Fig. 86 Control embryo.. 6000X.
Fig. 87 Embryo treated with 100 ug monocrotophos. 6000X. ifsuan.
I ll 1 1 2
Plate XXIX
Fig. 88-89 Notochord. Embryos injected at 3 days of incubation
and fixed 1 day later, er, endoplasmic reticulum;
n, nucleus; vac, vacuole.
Fig. 88 Control embryo. 5700X.
Fig. 89 Embryo treated with 100 ug monocrotophos. 5800X. 113
»?«■ 88a
,-. r . . ,; •-'.. ■*•." te r ,- •. r ' ■ V- <’ • ■• - . \ • •* l': :£> ‘. '•'?’ 'V/: . *. • v.?r »•* V*. A .'■ > .? v 7 . fc.. T** •w 1 • •", Jl. «*' > 7 V. t v
. y * S / -V lW - a - f iV.
ftS R g S 1 '-a'-
«*T*- * • • * 7* ^ r r w :'•* * & *. ’ •.** * / J & v <;•
*v>:' o v.» 4*' 'Vo* ’iv 5 .■+***>» * *s WCr , / ' • ** • •>> . w : U ■:■■■ ■ • • / C i Plate XXX
Fig. 90- 91 Myotome. Embryos injected a t 3 days of incubation
and fixed 1 day later, my, myofibril.
Fig. 90 Control embryo. 6700X.
Fig. 91 Embryo treated with 100 ug monocrotophos. 6700X.
Fig. 92- 93 Embryos injected at 3 days of incubation and fixed
lh days la te r. A, anterior end of embryo; size
bar = 500 urn.
Fig. 92 Control embryo.
Fig. 93 Embryo treated with 100 ug monocrotophos. Compression
of somitic areas is present. 911 116
Plate XXXI
Fig. 94- Embryos injected at 3 days of incubation and then fixed
lk days later, g'.spinal ganglia; m, muscle; n, notochord;
t, neural tube. 10 urn representative saggital sections.
Fig. 94 Control embryo. 85X.
Fig. 95 Embryo treated with 100 ug monocrotophos. Compressed
ganglia and myotome occur along with severely warped
notochord and neural tube. 85X.
Fig. 96- . Embryos injected at 3 days of incubation and fixed lk days
later, g, spinal ganglia; m, myotome; n, notochord;
s, sclerotome; t, neural tube. 1 urn cross sections.
Fig. 96 Control embryo. 580X.
Fig. 97 Embryo treated with 100 ug monocrotophos. 580X. 117
/»*
a 4 L * > J?il •**
r ( S S r
i J > '
,&£>r
* * *** • jte® *' > ^ v - V -> ^ . ,_ - * ■ ^
. ..» * » ? * ■ Plate XXXII
Fig. 98-99 Notochord. Embryos treated at 3 days of Incubation
and fixed lh days la te r, vac, vacuole.
Fig. 98 Control embryo. 6800X.
Fig. 99 Embryo treated with 100 ug monocrotophos. 6800X.
Fig. 100-101 Myotome. Embryos treated at 3 days o f incubation then
fixed IH days la te r, my, m yofibril.
Fig. 100 Control embryo. 6800X
Fig. 101 Embryo treated with 100 ug monocrotophos. 6800X. 119
-#< v** ■ *■"* 0 8
W.'i-r.r?
4.. V**
'•'. -• fe^V jV i
i s f l £ ® % s ®
iL'-.-l. Si'i*Li '.£ .) ' 1 2 0
. . Plate XXXIII
Fig. 102-103 Embryos injected at 3 days of incubation and fixed 2 days
la te r. A, anterior end of embryo, size bar = 500 urn.
Fig. 102 Control embryo.
Fig. 103 Embryo treated with 100 ug monocrotophos.
Fig. 104-105 Embryos injected a t 3 days of incubation and fixed 2 days
later, g, spinal ganglia; n, notochord, t, neural tube.
10 urn representative saggital sections.
Fig. 104 Control embryos. 85X.
Fig. 105 Embryos treated with 100 ug monocrotophos. 85X. IZT 122
Plate XXXIV
Fig. 106-107 Embryos injected a t 3 days of incubation and fixed 2h
days later, a, neural arch; g, spinal ganglia; n, notochord;
t, neural tube; v, vertebral centra. 10 urn representative
saggital sections.
Fig. 106 Control embryos. 85X.
Fig. 107 Embryos treated with 100 ug monocrotophos. Fused neural
arches and vertebral centra are present. 85X.
Fig. 108-109 Embryos injected at 3 days of incubation then fixed
3 days later, a, neural arch; g, spinal ganglia; n,
notochord; t, neural tube; v, vertebral centra. 10 urn
representative saggital sections.
Fig. 108 Control embryos. 85X.
Fig. 109 Embryos treated with 100 ug monocrotophos. Fused neural
arches and vertebral centra occur. The arrow indicates
the same position in the sections. Highly compressed
ganglia and vertebral centra are in this area and a
sharply curved notochord is also present. 85X. 123
1 0 6 a " 124
Plate XXXV
Fig. 110-111 Embryos injected at 3 days of incubation and fixed 5
days later, a, neural arch; g, spinal ganglia; m, muscle;
n, notochord; t, neural tube; v, vertebral centra.
Fig. 110 Control embryo. The 13 cervical vertebrae are numbered.
106X.
Fig. Ill Embryo treated with 100 ug monocrotophos. The neural
arches are fused, and the muscle less developed than
in the control. The first 12 cervical vertebrae are
numbered, and are severely compressed. The notochord
is still warped and the developing vertebrae are a
solid mass. The central canal of the neural tube is
almost straight. 106X 125
V i >2
P
ka
W 126
Plate XXXVI
Fig. 112-116 Stain for acetylcholinesterase with in vitro inhibitors.
Embryos were stained for AChE using the media of Karnovsky
and Roots (1964). Average stage 19+ (3 days of incubation),
ag, antonomic ganglia; g, spinal ganglia; m, myotome;
t, neural tube. 10 urn sections.
Fig. 112 Control. No inhibitor, ACH as substrate. 400X.
Fig. 113 Control. No inhibitor, no substrate. 400X.
Fig. 114 Sections treated with eserine, 130 ug (10"^M). ACH
as substrate. 400X.
Fig. 115 Sections treated with isoOMPA, 68 ug (10"5m) . ACH
as substrate. 400X.
Fig. 116 Sections treated with 284C51, 750 ug (7 x 10“^M).
ACH as substrate. 400X. US
/ m
X Y $ e. t, A
116 128
Plate XXXVII
Fig. 117-119 Stain for acetylcholinesterase with in vitro
inhibitors. Embryos stained for AChE using the media
of Karnovsky and Roots. Averagestage 19+ (3 days of
incubation), g, spinal ganglia; m, myotome; n, noto
chord; t, neural tube.10 urn cross sections.
Fig. 117 Control. No inhibitor. BuThCh as substrate. 400X.
Fig. 118 Sections treated with isoOMPA, 68 ug (HT5M). BuThCh
as substrate. 400X.
Fig. 119 Sections treated with 284C51, 110 ug (10"^M). BuThCh
as substrate. 400X.
Fig. 120-122 Stain for acetylcholinesterase using in. ovo inhibitors.
Embryos stained for AChE using the media of Karnovsky
and Roots. ACH as substrate. Embryos were treated at
3 days of incubation and fixed 3 hours la te r,
ag, autonomic ganglia; g, spinal ganglia; m, myotome;
n, notochord; t, neural tube. 10 urn cross sections.
Fig. 120 Control embryos. Positive reaction located in the
myotome, ganglia, and ventral horn of the spinal cord.
400X.
Fig. 121 Embryos treated with 200 ug isoOMPA. 400X.
Fig. 122 Embryos treated with 300 ug of 284C51. 400X. 129
*s-» 130
Plate XXXVIII
Fig. 123 Stain for acetylcholinesterase. Embryo injected with
100 ug monocrotophos a t 3 days of incubation and fixed
3 hours later, g, spinal ganglia; m, myotome; n,. noto
chord; t, neural tube. 10 urn cross sections. 400X.
Fig. 124-129 Embryos injected at 3 days of incubation and fixed in FAA
3 days later. Cartilageous skeleton was stained using
the method of Ojeda, et al_. (1970).
Fig. 124 Embryo treated with 100 ug monocrotophos. 10X.
Fig. 125 Embryo treated with 100 ug isoOMPA. 10X
Fig. 126 Embryo treated with 130 ug eserine. 10X.
Fig. 127 Embryo treated with 300 ug 284C51. 10X.
Fig. 128 Embryo treated with 1 mg 284C51. 10X.
Fig. 129 Control embryo. 10X. 131 132
Plate XXXIX
Fig. 130-135 Embryos fixed a t 6 days of incubation in FAA and
stained using the method of Ojeda e t aK (1970).
Fig. 130 Control embryo. 10X.
Fig. 131-132 Embryos treated with 100 ug monocrotophos at 3 days
of incubation. 10X.
Fig. 133-135 Embryos treated with both monocrotophos (100 ug) and
2-PAM (1 mg) at 3 days of incubation. 10X. 133 Plate XL
Fig. 136-139 Embryos fixed at 6 days of incubation in FAA and
stained using the methods of Ojeda ert al_. (1970).
Fig. 136-137 Embryos treated with 2-PAM (1 mg) at 2 days of
incubation and then with monocrotophos (100 ug) plus
2-PAM (1 mg) at 3 days of incubation. 10X.
Fig. 138-139 Embryos treated with monocrotophos (100 ug) plus
2-PAM (1 mg) at 3 days of incubation then with 2-PAM
(1 mg) a t 4 days of incubation. 10X.
Fig. 140-141 Embryos injected a t 3 days of incubation and fixed
3 hours later. Sections stained for AChE using the
media of Karnovsky and Roots (1964). ag, autonomic
ganglia; g, spinal ganglia; m, myotome; n, notochord;
t, neural tube. 10 urn sections.
Fig. 140 Embryos treated with monocrotophos (100 ug) plus
2-PAM (1 mg). 400X.
Fig. 141 Control embryo (same as Fig. 120). 400X. 135 VMM*' Vita
Christina Irene Lusk was born on November 2, 1948 in Starkville,
Mississippi. She was graduated from Starkville High School in
May 1966. She attended Mississippi State University, receiving her
B.S. in Zoology in May 1970 and her M.S. in Zoology in January 1972.
From 1972 to 1974, she was employed as Senior Laboratory Technician,
Pesticide Laboratory, Entomology Department, University of Kentucky,
Lexington, Kentucky.
She is currently a candidate for the degree of Doctor of
Philosophy in Zoology in the Department of Zoology and Physiology,
Louisiana State University, Baton Rouge, Louisiana.
136 EXAMINATION AND THESIS REPORT
Candidate: C h ristin a Ire n e Lusk
Major Field: Zoology
Title of Thesis: Development of the Cervical Region of Chicken Embryos Studied via the Teratogenic Effects of Monocrotophos
Approved:
^M &jor Professor and Chairman
Dean of the Graduate School
EXAMINING COMMITTEE:
JIO ------
Date of Examination:
1° \ ^ , l v