/ . Embryol. exp. Morph., Vol. 12, Part 3,pp. 457-463, September 1964
Printed in Great Britain
The transfer of coelomic eggs between frogs
by LORA HARRER LAVIN 1
From the Department of Zoology, University of Michigan
IN the analysis of events immediately preceding and following fertilization of
the frog egg, it is frequently desirable to treat the eggs with various reagents.
Any such treatment must circumvent the barrier represented by the fertilization membrane and the jelly layers. These membranes can be removed from fertilized eggs mechanically or by treatment with proteolytic enzymes (Spiegel, 1951)
without impairing development. However, unfertilized eggs from which the
jelly has been removed mechanically cannot be fertilized by sperm unless the
jelly is restored, and chemical removal of the jelly also inhibits their ability to be
parthenogenically activated (Barch & Shaver, 1959). Therefore an alternative
method for obtaining jelly-free eggs for treatment prior to fertilization is
necessary.
Coelomic eggs are invested by a vitelline membrane, but they are free of jelly
layers and the fertilization membrane has not yet been formed. This recommends
them as the stage for exposure to reagents whose effects on fertilization are to be
analyzed. However, although Subtelny & Bradt (1960) review circumstances
under which coelomic eggs may be activated parthenogenically and develop normally, normal development is not obtained after insemination with sperm (Rugh,
1962). Apparently, normal fertilization of many eggs can only be obtained after
eggs have passed through the oviduct. It therefore seemed important to determine
whether or not normal fertilization could be obtained using coelomic eggs removed from a female, treated with various reagents, then returned to a host
female where they could pass through the oviduct prior to insemination.
Arnold & Shaver (1962), Humphries (1956) and Subtelny & Bradt (1960)
obtained coelomic eggs by ligation or removal of the oviducts of donor animals
prior to pituitary stimulation. Arnold & Shaver (1962), Humphries (1956) and
Rugh (1962) transferred such eggs into host females through an incision made
in the body cavity of anesthetized animals. Rugh (1962) reported that eggs
transplanted to sexually inactive females will pass through the oviducts and
collect jelly. In this case, all uterine eggs are of donor origin. However, jelly
secretion is not normal and the percentage of eggs fertilized is low. Normal
results are only obtained if sexually active hosts are used. To identify the donor
1
Author's address: 4306 Miller Road, Wilmington, Delaware 19802, U.S.A.
458
LORA HARRER LAVIN
eggs, Arnold & Shaver (1962) and Aplington (1957) used ovariectomized hosts
while Humphries (1956) distinguished donor eggs by staining them.
The above investigators have shown that eggs transferred between females
can develop normally. However, their operative techniques are tedious for the
investigator and traumatic to the animals. The simple technique described here
was used in a series of experiments which tested the effects of antisera on frog
egg fertilization (Nace & Lavin, 1963; Lavin, 1963) but can also be applied to
other types of experiments. It avoids the necessity of complex operative techniques and can be used routinely.
MATERIALS AND METHODS
Rana pipiens, obtained from C. H. Mumley, Alburg, Vermont, were induced
to ovulate during the winter by injecting 5 mg. of progesterone (Sigma Chemical
Company; suspended in corn oil at a concentration of 25 mg./ml.) into the dorsal
lymph sac and one female pituitary gland into the coelomic cavity (Wright &
Flathers, 1961). In the spring the progesterone was omitted. One donor and
one or more host frogs were injected for each experiment.
The frogs were kept at 18°C. and, starting 12 hr. after injection, the donor was
examined periodically to determine when the first eggs could be expressed from
the uterus. Depending on the season, this occurred from 12 to 22 hr. after
injection. Two or three hours later the donor frog was pithed, and ovulated eggs,
which had not yet entered the oviducts, were collected from the coelom with a
transfer pipette. Since coelomic eggs are exposed to an isotonic environment,
they were washed with several changes of standard Holtfreter's solution and
were stored in this medium.
The eggs were counted and placed in small 'baskets' made by stretching a
swatch of raw silk over one end of a glass ring, where the silk was held by a closely
fitting plastic cuff. Such basketsfilledand drained rapidly as they were transferred
between solutions and thus made possible uniform exposure of all eggs to the
reagents. Usually, 100 eggs were placed in each basket.
After washing, the eggs were exposed to the experimental and control reagents.
They were then further washed, transferred to a solution consisting of 0-1 g. of
neutral red in 100 ml. of standard Holtfreter's solution for 1 min., washed free
of excess dye and drawn into a 5-ml. syringe. The eggs were injected into the body
cavities of host females by gently ejecting them from the syringe fitted with a large
internal diameter syringe needle 4 cm. long. The narrowest passage in this system
was 2 mm., which readily accommodated the passage of R. pipiens eggs. The
sharpened needle was inserted at a 45 degree angle to the body wall at a point
about 1 cm. caudal to the sternum and slightly to the right or left of the ventral
abdominal vein. With the syringe held vertically, the eggs settled toward the
needle and usually not more than 1 ml. of Holtfreter's solution was injected
with the eggs.
Transfer ofcoelomic eggs between frogs
459
The host frogs were placed in separate labeled containers and were kept at
18°C. for at least 16 hr. during which the eggs passed through the oviducts and
were allowed a sufficient period of residence in the uterus (Rugh, 1962).
Insemination, using sperm suspensions pooled from one or male frogs, was
carried out as described by Rugh (1962). The transplanted eggs were readily
identified by their light red color. They were separated from the host eggs
starting 60 min. after insemination when rotation had taken place. First, all
unrotated donor eggs were removed. The egg masses were then inverted and
sufficient time allowed for the eggs to reorient themselves. Again, all unrotated
colored eggs were removed. This procedure was repeated once more. The remaining colored eggs were considered as activated and were isolated. The
developmental fate of the activated eggs was closely followed, at least through
the gastrula or tail bud stages. Similarly isolated host eggs were retained as
controls for the potency of the sperm preparations.
RESULTS AND DISCUSSION
Collection ofcoelomic eggs
Since R. pipiens ovaries release several thousand eggs over a period of several
hours, large numbers of coelomic eggs could be obtained from a single frog
without prior surgical treatment if they were collected at the proper time in the
ovulation cycle. This time varied from 14 to 24 hr. after hormone injection, depending on the batch of frogs, time of year and type and amount of hormone administered. However, for any combination of variables, eggs could be found in
the coelom from 1 to 4 hr. after the first eggs could be expressed from the cloaca.
Alternatively, jelly-free eggs could be obtained by stimulating ovulation in
vitro (Wright, 1945; Nadamitsu, 1953). The experiments of Nadamitsu (1953)
show that such eggs are capable of normal development after transfer to hosts.
However, in this laboratory the results obtained by in vitro ovulation have been
less satisfactory for routine use than the method described above. This may not
be true for other amphibians.
Recovery after transfer
In over seventy-five transfers of 100 eggs per host female, recovery has ranged
from 29 to 100 per cent. Table 1 shows representative results obtained with eggs
used as controls to test the effects of various antisera on fertilization and development. These control eggs were exposed to standard Holtfreter's solution.
Additional data on control eggs and data on experimental eggs are presented
elsewhere (Nace & Lavin, 1963; Lavin, 1963).
It is evident from Table 1 that never were more stained eggs recovered than
were injected. Though the results suggested that stain was not transferred from
460
LORA HARRER LAVIN
donor to host eggs in vivo, this possibility was tested by keeping stained and unstained eggs in close contact in vitro overnight. There was no evidence of any
transfer of the stain.
In some cases, less than 100 per cent recovery could perhaps be attributed to
destaining or failure to recognize lightly stained eggs. However, this probably
did not constitute a major source of error since stained eggs retained their color
in vitro for many hours. On occasion dissection of the host frogs showed debris
which could be attributed to damaged and disintegrated eggs. However, more
frequently the poor recovery could be attributed to failure to express all the eggs
from the uterus.
Development after transfer
Transplanted eggs were capable of normal development after insemination.
In the examples summarized in Table 1, activation, as defined by rotation, ranged
from 69 to 97 per cent. In normal fertilization, activation averaged 95 per cent.
The cases of low percentage activation must be examined in the context of the
several steps in the process which offered opportunity for damage to the eggs.
Neither the 'basket transfer' of eggs through the solutions nor the passage of
eggs through the injection needle produced gross macroscopic damage, though
the possibility of microscopic damage was not subjected to critical evaluation.
Staining with neutral red could be eliminated as a possible source of damage,
because of the high percentage of development in the optimal experiments.
Humphries (1956) also stained coelomic eggs with 0-1 per cent neutral red
without harming them. Thus, it seems most reasonable to attribute the cases of
poor activation to biological variations among the batches of eggs.
The cleaved eggs almost invariably developed to late gastrula and continued
to develop into normal tadpoles in cases where extended observations were made.
Within limits, the maintenance of coelomic eggs in vitro did not affect their
capacity for normal development after transfer. In most cases, the first polar
body had not yet been extruded when coelomic eggs were collected. However,
their stage of maturation did range from breakdown of the germinal vesicle to
metaphase II. All the eggs progressed to metaphase II within 8 hr. if kept in
standard Holtfreter's solution at 18 to 26°C.
Within about 24 hr. changes began to occur in the distribution of the surface
pigment (Holtfreter, 1943). Following these changes, the eggs eventually degenerated. In spite of these changes eggs have been maintained in vitro for up to
24 hr. before transfer and, after recovery and insemination, have developed
normally.
Experiment 6 (Table 1) illustrates the suitability of this egg-transfer technique
for testing the effect of various manipulations on fertilization and development.
In this experiment eggs from the same batch were injected into two different hosts.
No significant differences in the proportions of activation and development
were observed.
1
2
3
4
5
6
Experiment
number
63
71
57
66
38
68
60
71
73
78
82
55
79
74
nvtst
No.
U
isumoer ana
% of eggs
recovered*
AT
* 100 eggs transferred in each case.
72
86
62
43
55
72
61
51
63
48
35
30
57
45
89
97
73
80
69
86
81
81
89
84
53
79
84
75
activated
recovered
No.
%ofno.
%ofno.
Cleavage
% of no.
recovered
Activation
51
61
45
34
28
57
42
No.
1
Development of normal eggs transferred between female Rana pipiens
TABLE
72
84
58
41
51
72
57
recovered
%ofno.
A
100
97
94
97
93
100
93
cleaved
%ofno.
Gastrulation {Stage 12)
2-
a
a
a
3*
'nfr ogs
462
LORA HARRER LAVIN
SUMMARY AND CONCLUSIONS
The use of jelly-free amphibian eggs for certain types of experiments on fertilization and development is often desirable. The jelly layers and fertilization
membrane can be easily removed by chemical techniques without damage to
fertilized eggs. However, unfertilized eggs from which the jelly has been removed cannot be fertilized. Coelomic eggs can be conveniently used for experiments designed to test the effects of various treatments on fertilizability provided
they are made fertilizable after treatment by passing them through the oviduct of
host females. The method for collection and transfer of coelomic eggs described,
offers several advantages over those previously described. Frogs are stimulated
to ovulate. Soon after the appearance of the first extrudable uterine egg, the
animals are opened and eggs free in the coelom are collected, transferred through
the various experimental and control solutions, stained and reintroduced into
ovulating host females by injection from a syringe through a large internal diameter needle inserted into the body cavity. This simple method avoids the use
of anesthetic and surgical techniques which traumatize the frogs and mitigate
against uniformity of results. Twenty-nine to 100 per cent of injected eggs were
recovered and 41 to 84 per cent developed normally to gastrula or later stages.
The method may be used routinely, even by the inexperienced investigator.
RESUME
Transfert d'ceufs ccelomiques entre grenouilles
L'utilisation d'ceufs d'Amphibiens depourvus de gangue est souvent desirable
pour certains types d'experiences sur la fecondation et le developpement. L'usage
de techniques mecaniques et chimiques pour deganguer les oeufs extraits des uterus
ne s'est pas revele satisfaisant. Les oeufs coelomiques conviennent a ces experiences a condition qu'on puisse les rendre fecondables en les faisant passer a
travers l'oviducte de femelles receptrices. On decrit une methode de recolte et de
transfert d'ceufs coelomiques, qui offre plusieurs avantages sur celles qui ont ete
decrites auparavant. On stimule l'ovulation des grenouilles. Peu apres l'apparition du premier oeuf uterin expulsable, les animaux sont ouverts et on
recolte les oeufs libres dans le coelome. Ces oeufs sont transported dans diverses
solutions experimentales et temoins, colores et reintroduits dans les femelles
receptrices en cours d'ovulation, par injection a l'aide d'une seringue. Cette
methode simple evite l'utilisation de techniques anesthesiques et chirurgicales
qui traumatisent les grenouilles et perturbent Puniformite des resultats. Vingtneuf a 100% des oeufs injectes ont ete recuperes, 41 a 84% se sont developpes
normalement jusqu'a la gastrula ou des stades ulterieurs. La methode peut etre
utilisee couramment, meme par un chercheur inexperimente.
Transfer of coelomic eggs between frogs
463
ACKNOWLEDGEMENTS
This research was done in partial fulfillment of the requirements for the degree of Doctor
of Philosophy. It was supported in part by grants from the U.S. Public Health Service: a
National Institutes of Health Research Grant (GM 05409) to Dr Nace and an NIH Graduate
Training Grant (GM 989). The author would like to thank Dr George W. Nace for his advice
during the conduct of this investigation.
Please address reprint requests to Dr Nace, Department of Zoology, University of Michigan,
Ann Arbor 48104, U.S.A.
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{Manuscript received 22nd February 1964)