/. Embryo!. exp. Morph. Vol. 27, l,pp. 103-120, 1972
Printed in Great Britain
103
Vitamin E as an extrinsic
and intrinsic signal controlling development in the
rotifer Asplanchna: uptake, transmission and
localization of [3H]oc-tocopherol
By C. W. BIRKY, Jr.1 AND JOHN. J. GILBERT 2
From the Department of Biological Sciences,
Dartmouth College, Hanover
SUMMARY
An increase in the level of vitamin E in the diet of saccate, amictic female Asplanchna
modifies the development of their parthenogenetic female embryos developing in utero. The
offspring develop prominent outgrowths of the body wall (BWO response), and some mature
as mictic females which produce male progeny by haploid parthenogenesis. We have followed
the fate of [3H]a-tocopherol fed to immature females. In each generation, females lose about
50% of their a-tocopherol; the remaining material, which is 76-100 % undegraded, is
transmitted almost entirely to their male or female offspring. The a-tocopherol content of a
female is proportional to the degree of her BWO response. The results support the hypotheses
that vitamin E acts directly on embryos to control development, i.e. that it serves as both an
intrinsic and extrinsic control signal.
INTRODUCTION
Organisms make extensive use of extrinsic (environmental) signals, as well as
intrinsic signals, for the modification and control of development. The response
to extrinsic signals is nowhere more strikingly seen than in the seasonal changes
in morphology and mode of reproduction shown by many groups of freshwater
planktonic invertebrates. Seasonal changes in morphology (cyclomorphosis)
are especially common among the rotifers and Cladocera, where they are often
sufficiently drastic to create taxonomic confusion. Seasonal transitions between
asexual (parthenogenetic) and sexual reproduction are also characteristic of
these groups. These changes in morphology, physiology, and mode of reproduction, which we designate collectively as developmental polymorphism, have
in many instances been shown to be controlled by environmental stimuli (see
review by Hutchinson, 1967). We have been using the monogonont rotifer
1
Author's address: Faculty of Genetics, The Ohio State University, Columbus, Ohio
43210, U.S.A. Reprint requests should be sent to this address.
2
Author's address: Department of Biological Sciences, Dartmouth College, Hanover,
N.H. 03755, U.S.A.
104
C. W. BIRKY, JR. AND J. J. GILBERT
Asplanchna for a detailed study of this phenomenon. In particular, we are
concerned with the nature of the effective environmental stimuli (Birky, 1964,
1969; Kiechle & Buchner, 1966; Gilbert, 1967,1968,1969; Gilbert & Thompson,
1968; Buchner, Kiechle & Tiefenbacher, 1969), the mode of action of these
stimuli at the cellular level (Birky, 1968), and the adaptive significance of
the response to these extrinsic signals (Gilbert & Thompson, 1968; Birky,
1969).
Natural populations of A. brightwelli and A. sieboldi, and laboratory populations grown on a diet of paramecia, consist mainly or entirely of females which
are saccate (sack-shaped) and amictic (producing clones of genetically identical
daughters by diploid parthenogenesis). In natural populations at various times
during the summer, the saccate females (especially of A. sieboldi) produce
daughters with body-wall outgrowths (BWO's, or humps; see Birky & Power
(1969) for diagrams of various morphotypes). These daughters are often mictic
females, which produce males by haploid parthenogenesis or, if fertilized,
diploid resting eggs which hatch as amictic females. In the laboratory, the
production of daughters with BWO's and of mictic daughters may be induced
at will by the addition of vitamin E (a-tocopherol) to the maternal diet (Gilbert
& Thompson, 1968). This treatment also increases the number of nuclei in
certain organs and decreases the longevity and fecundity of females (Birky &
Power, 1969; Birky, 1969). The level of vitamin E in the maternal diet constitutes
an extrinsic signal which, directly or indirectly, acts on embryos in utero to
control morphogenetic growth of the body wall (BWO response), the mitosis/
meiosis alternative in the oocytes (mictic female response), and the number of
mitotic divisions in certain embryonic organ Anlagen (Birky, 1968).
In this paper, we ask whether or not vitamin E acts directly on the embryos to
control development, i.e. whether or not it also serves as an intrinsic signal or
inducer. A direct test, using embryos reared in vitro, proved impractical. However, Birky & Power (1969) have shown that, when amictic females are fed
vitamin E during their immature period only (Ax generation), the BWO response
is seen not only in their daughters (A2 generation), but also in at least two subsequent generations (A3 and A4) when the initial stimulus is sufficiently strong.
The magnitude of the response typically remains constant or increases in the
A3 females, then declines in the A4 and A5 generations. If vitamin E itself is the
intrinsic inducer, then it must be transmitted intact with high efficiency from
generation to generation, with a gradual dilution of effective concentration. We
have therefore examined the pattern of uptake, transmission, and loss of labelled
a-tocopherol; our data show that it is indeed transmitted intact from parent to
offspring with high efficiency, and that the a-tocopherol content of a female is at
least roughly proportional to the magnitude of her BWO response. Our results
also are consistent with the hypothesis that vitamin E is required in these rotifers
for male fertility. This hypothesis provides an evolutionary rationale for the
use of dietary vitamin E as an extrinsic signal for sexual reproduction.
Control of development by vitamin E
105
MATERIALS AND METHODS
Asplanchna brightwelli, inbred clone 5B4S76 (Birky, 1967), was used in one
experiment. All other work used A. sieboldi of clone 12C1, which is highly
sensitive to dietary vitamin E (Birky, 1969). The animals were kept in plastic
dishes or depression slides at 25 °C in the dark. The culture medium (GCF)
is a dilute infusion of Perth grass containing Paramecium aurelia (with some
Aerobacter aerogenes and miscellaneous contaminating bacteria) (Gilbert,
1968). Penicillin was added in some cases to reduce contamination. Sterile GCF
for washing animals was obtained by filtering GCF through paper and subsequently through a Millipore filter. The population density was generally 10
females/ml, with all animals being transferred to fresh medium every one or
two days. Under these conditions, the parthenogenetic generation time is about
1-1 £ days.
Labelling experiments
Initial labelling experiments used [14C]^-a-tocopheryl succinate (Distillation
Products Industries, sp.act. 1-0616 mCi/mM); subsequently we used [3H]dl-octocopherol from Amersham/Serle, with a specific activity of 2500 or 2720
mCi/mM and an advertised purity of 97-98 %. Both molecules were labelled in
the 5-methyl group; specific activities are those quoted by the manufacturers.
The [3H]a-tocopherol was stored in benzene: ethanol under N 2 at 4 °C; aliquots
were dried and taken up in ethanol as needed for experiments. The [14C]atocopherol was stored in ethanol under air at 4 °C and may have undergone
some oxidation before use. Labelled and unlabelled a-tocopherol in ethanol
were injected with a syringe directly into the culture fluid to form a fine emulsion
at a final concentration of 10~6 M (occasionally, 10~7 M). This emulsion is ingested
by paramecia and subsequently by the rotifers.
For scintillation counting, labelled animals were washed with sterile GCF to
remove all unbound radioactivity, rinsed in distilled water, and transferred
with a minimum of fluid to Bray's solution. Radioactivity is completely extracted
in Bray's; treatment with Biosolve (Eastman Organic), sonication, homogenization, and/or extraction with methanol alone or with N-hexane released no
significant additional amount of radioactivity. Sample vials contained from 1
to 500 rotifers and were counted for from 10 to 100 min as needed to obtain
significant counts, in a Nuclear Chicago 6801 scintillation counter using
quenched standards and the channels ratio method to determine efficiencies.
The results are expressed in net dpm/female and in moles/female (using manufacturers' stated activities) to correct for variations in counting efficiency, but
our conclusions are unchanged if calculations are made on the basis of net
cpm/female. Background counts (generally about 27 cpm with 5 % S.D. for 14C
and about 31 cpm with 6 % S.D. for 3H) were obtained from matching vials
containing rotifers treated identically to the experimental animals, but with
unlabelled ^/-a-tocopherol. Where appropriate, background counts from a set of
106
C. W. BIRKY, JR. AND J. J. GILBERT
control vials were averaged and the mean was used to correct a number of
different experimental vials. All counts were significantly above background
unless noted otherwise.
Mass culture:
clone of amictic
females in G.C.F.
Con
A'I
I
Score
Bray's
Score
Discard
G.C.F.
24 h
I
24 h
24 h
Separate, score, and count A'j and A) as above
Fig. 1. Flow-sheet diagram of a typical experiment to determine the uptake, loss, and
transmission patterns of a-tocopherol in Asplanchna. See text for explanation.
The design of a typical pulse-labelling experiment is shown in Fig. 1. Parthenogenetic generations are called Al5 A2, etc. Immature A± females aged 0-5 h are
collected from mothers (Ao) reared in the absence of added vitamin E. They are
allowed to mature in GCF plus a-tocopherol for about 20 h, at which time their
first offspring are nearly ready for birth. The A± females are washed free of
unbound radioactivity in sterile GCF; some are then placed in Bray's solution
for counting and some are put in GCF to obtain the next generation. Between 12
Control of development by vitamin E
107
and 24 h later, these animals are separated from their A2 offspring; the old (Aj)
mothers are either put in Bray's or are kept in GCF to obtain their later A2
offspring. Of the immature A2 females, which are generally of parity (birth
order) I, II, and III, some may be placed in GCF to obtain the A3 generation
and some are put in Bray's for counting.
In some experiments, the response of the animals to a-tocopherol was
measured using the morphotypic score system of Birky & Power (1969). A
sample of animals is killed in 30 % ethanol and expanded in 70 % ethanol, and
the development of the body-wall outgrowths (BWO's or humps) in each
animal is scored on an ordinal scale ranging from 1 (saccate) to 5 (fully humped).
The mean score of a group of experimental animals is compared to that of the
corresponding group of controls reared in GCF without added vitamin E.
Thin-layer chromatography of extracts
In initial experiments, we found that pure [3H]a-tocopherol at the very low
concentrations used in these experiments, as well as labelled material extracted
from females, underwent rapid oxidation during chromatography, with as
much as 96 % of the a-tocopherol being converted to a-tocopheryl quinone and
other compounds. This problem could be overcome partially by minimizing
exposure to light and air and especially by the addition of large amounts of
cold carrier a-tocopherol to the extraction mixture. The final procedure adopted
began with extraction of females in 1 ml re-distilled acetone containing 0-1 mg
each of J/-a-tocopherol and a-tocopheryl quinone, under N 2 at 4 °C for 1 h.
The acetone was removed and the animals, after being rinsed in 1 ml fresh
acetone, were dried in a vial for counting. From the pooled extracts, an 0-2 ml
sample was taken and also dried for counting; the remaining extract was dried
under a stream of N 2 , taken up in chloroform, and spotted on plates with 250
jam layers of silica gel G. Adjacent channels on each plate were used for the
extracts of labelled females, extracts of unlabelled control females (for background counts), and extracts of unlabelled females to which had been added, at
the beginning of extraction, a sample of [3H]a-tocopherol of approximately the
same activity as the labelled females (to determine the amount of oxidation
occurring during the procedure).
All plates were developed in chloroform under N 2 in dim light, and then
sprayed with I 2 vapor for approximate localization of the positions of atocopherol and a-tocopheryl quinone. Each channel was divided into 16 to 18
segments of about 1 cm each, which were then scraped off into individual vials.
Scrapings, extract aliquots, and extracted females were counted in toluenePPO-POPOP fluid. Counts of dried animals and of extract aliquots were used
to calculate the efficiency of extraction and of recovery of material from the
plates. Extraction of label ranged from 2 to 95 %, with most values 89 % or
greater; the low values are almost certainly in error due to difficulties in handling
the animals and extracts. Calculated recovery ranged from 50 to 181 %. Neither
108
C. W. BIRKY, JR. AND J. J. GILBERT
extraction efficiency nor recovery showed any correlation with the calculated
percentages of a-tocopherol in the animals, and probably introduced no significant bias into the results.
The d/-a-tocopherol and a-tocopheryl quinone used in these experiments were
purchased from Pierce Chemical and Nutritional Biochemicals Corporation,
respectively. Three dimeric oxidation products of a-tocopherol (the spiro
dimer; 5,5'-Bi-a-tocopherol; and 5,5'-methylene bis-(y-tocopherol)), and tocopheronolacteone were donated by Distillation Products Industries through the
kindness of Dr David C. Herting.
Autoradiography of labelled females
Following conventional fixation schedules in osmic acid or glutaraldehyde,
labelled a-tocopherol is rapidly extracted from tissues during dehydration in
ethanol before embedding in methacrylate, or during embedding in the watersoluble medium 'Polyamph' (Polysciences, Inc.). This difficulty was overcome,
at least for some blocks, by fixing washed animals in 2 % osmic acid for 17 h
at 4 °C, washing, and post-fixing in 0-5 % KMnO 4 for 15 min. Fixatives were
made in 50 % Gilbert's (1963) saline. After careful rinsing, the animals were
dehydrated rapidly in ethanol and embedded in methacrylate. Two-micron
sections were covered with Kodak NTB 3 emulsion and exposed for 2\ months.
Only about 3 % of the total label was extracted during dehydration.
RESULTS
Pulse and long-term labelling of Ax females
The results of the first labelling experiments are shown in Table 1. Females
from a culture exposed to [14C]a-tocopherol continuously for 6 days, including
animals from five or six successive amictic generations, contained about 5-4 x
Table 1. Incorporation of[uC]oc-tocopherolby Asplanchna sieboldi females
Exp.
Time*
Females
dpm±s.D. /females
Moles/female
1
2
3
6 days
24 h
24 h
4
20 h
A^Ae
A1
A1
Ax
AiKCN-killed
42-3 ±3-4/50
9-0±2-5/50
94-8 ±3-9/260
359-6 ±6-5/500
28-6 ±2-8/110
5-44 xlO~ 1 3
106 xlO" 1 3
2-50 x 10~13
4-76 xlO" 1 3
1-70X10"13
* In 10-6M[14C]d-a-tocopherol.
10~13 moles of a-tocopherol per female. (This assumes that all label is in atocopherol; see below.) Ax females labelled during their immature period only
(20-24 h) contained from 1-1 to 4-8 x 10~13 moles/female. These results with
[14C]a-tocopherol are consistent with those obtained with [3H]a-tocopherol
(Tables 2-4).
Control of development by vitamin E
109
The incorporation by Ax females varied greatly between experiments. The
range was 11-6-753 x 10~15 moles/female, or, excluding the unusually low value
in Exp. 10, 106-753 x 10~15 moles/female. A statistical comparison of counts of
individual Ax females from Exps. 9 and 11 showed that the difference in mean
Ax uptake was highly significant, and cannot be accounted for by sampling error
or by differences in background counts. We conclude that these differences
represent real variability in uptake by females. Such variability is not surprising,
as it is completely consonant with biological variability in Asplanchna measured
by other means, e.g. (1) mictic female production induced by a-tocopherol
(Gilbert & Thompson, 1968); (2) reproductive parameters (C. W. Birky &
J. J. Gilbert, unpublished); and (3) sensitivity to streptomycin (C. W. Birky &
A. E. Brodie, unpublished).
Label in females exposed to [14C]a-tocopherol continuously for 6 days (Table
1, Exp. 1) fell well within the range of label in females exposed to 20- to 24-h
pulses of [14C] or [3H]a-tocopherol. This result agrees with previous data on the
morphological response to vitamin E (Birky & Power, 1969) and suggests that
the animals may be essentially ' saturated' with a-tocopherol within 24 h or less.
It was expected that at least a portion of this label might be adsorbed on the
surface of the animals rather than ingested. This was verified by killing females
with KCN and placing them in GCF plus [14C]a-tocopherol for 20 h. The corpses
contained approximately one-third as much radioactivity as did living females.
We have not attempted to use this value to correct our Ax data for surface
adsorption, because it is unlikely that living animals have precisely the same
adsorption properties as corpses.
In a later experiment (Exp. 9) using [3H]a-tocopherol of very high specific
activity, individual Ax females were counted to obtain information about the
individual variation in uptake. Counts of ten females labelled for 20 h with
10~6 M [3H]a-tocopherol showed a range of 2-84-11-32 x 10~13 moles/female,
with a mean of 7-53 x 10~13 moles/female. Individual Ax females thus show a
fourfold variation in uptake of vitamin E; it is not known how much of this
variability is in actual ingestion and how much is in adsorption.
Fate of ingested cc-tocopherol: patterns of loss and transmission
Table 2 shows the data from seven experiments concerning the loss of labelled
a-tocopherol and its transmission from generation to generation. It should be
noted first that, in contrast to the almost 70-fold variation in Ax uptake, the
percentage of label which is transferred from generation to generation is
relatively consistent from experiment to experiment. The following pattern
emerges for A. sieboldi (Exps. 7, 9, 10, 11 and 12):
(1) A2 females transfer 14-5-28-7 % of their total label to their first-born
offspring (A3I females). Succeeding A3 offspring (parities II-IV) receive successively smaller portions of the total parental label. A 3 males (Exp. 7) each receive
smaller proportions of parental label (10-2 %) than do females.
10
9b
9a
Exp.
A,
Ai
A!
A3
A,
A..
Ao
Ai
A,
A,
Ao
A1
Ai
Ao
Ao
A3
Parity
Mixed
Mixed
I
I
I + II
1 + 11
Mixed
Mixed
I
11
III
IV
V
VI
Mixed
Mixed
I + II
l + II
III
1
Mixed
I
Generation
337
262
11-4
716
3-28
116
753
62-2
117
112
56-8
190
19-8
500
753
144
54-6
65-3
51-9
16-6
11-6
211
15
—
—
—
—
—
—
—
—
—
—
—.
—
4-5(1-3)
3-7(1-6)
4 0 (1-7)
3-2 (1-8)
3-0(1-5)
2-5 (—)
MeanJJ
morph
scores
neratio n
A,
Parity
Mixed
Mixed
Ai
A,
1
A2'
1
Ao
II
A2
111
A2
IV
Ag
IV
I
A3
II
A3
III
A3
IV
A3
I + I I + III + IV
A3
Mixed
A,
12H
Mixed
Ai
A2
I + II
Mixed
A;
12L
Mixed
l+II
A2
Mixed
Ai
Ab
Mixed
Ai
I+II
A2A
AoA
1-4- II
1
A3
I+II
AjJM
I + II
A-, c?
I + Il + l I I + l V + V
or A. brightwelli (Exp. Ab).
11
Exp.
dpm
5-03ft
679 X
103f
82-9
7-71
67-8f
56-4f
33-9
602
18-9
907
7-34
4-62
A
112
171
13-7
1-28
11-2
9-34
5-62
100
3-14
1-50
1-21
0-77
O-83§
298
113
48-5
233
431
22-3
672
266
51-8
13-8
3-96
351
16-6
7-67
Moles x 10-
Label per ? or S
1795
684
293
1470
260
134
4060
1605
312
83-5
23-9
212
100
101
* A. siebolcli (Exps. 7, 9, 10, 11, 12)
t Mean of two samples.
j , §, II. "! Mean of individual counts of 10, 9, 6, or 3 females, respectively.
I t Values uncertain because counts very low. A 3 females from parity IV A2 mothers.
** Animals counted after birth of some A 3 daughters and loss of some label.
XX In parentheses, scores of controls without added a-tocopherol.
Moles x 10-
dpm
1870
1456
63-4
39-7
18-2
6-46
4170J
345§
649§
620§
315§
105§
110||
27-711
4170
800
303**
362
282
921
64-6
11-7
Label per $ or 3
15
—
—
—
—
3-4 (2-3)
.
—
—
—
4-9 (30)
1-7(1-9)
—
3-4 (2-5)
—
3-7(2-1)
2-4 (2-2)
3-3 (2-2)
—
3-3(1-7)
2-6(1-7)
2-9 (2-0)
2-7(1-9)
2-3 (2-0)
MeanJJ
morph
scores
Table 2. Incorporation and transmission of[3H]dl-a-tocopherol by Asplanchna* females. Ax females labelled 18-21-5 h
in 10~6 or 10~7 M a-tocopherol
m
w
s
O
bd
Control of development by vitamin E
111
(2) Ax females transfer smaller proportions (3-4-16-3 %) of their total label
to their A2I offspring; again, subsequent offspring receive successively smaller
portions of the parental label. The interpretation of the kx-K2 data is complicated by the probability that some of the Ax label represents material adsorbed
on the surface of the females. This material might well contribute to the loss of
label (discussed below), by being washed off as the animals swim or by being
oxidized by the bacteria which also adsorb to the animals' surfaces, but it
could not be transferred to their daughters. It may be that, in Exps. 7 and 11,
which show a markedly lower transfer from Ax to A2 than from A2 to A3, the Ax
females had a relatively high proportion of surface-bound label. In Exp. 9a,
+
Ut
*
'
Fig. 2. Autoradiograph of A1 female A. sieboldi fed 10~ 6 M [3H]a-tocopherol for 20 h
after birth. Note intensive label in uterine cavity (ut), and absence of label in
pseudocoel (pc) and stomach cavity (stc). There is some label in the stomach wall
(stw), obscured by heavily stained lipid droplets. Focus is a compromise between
the levels of the tissue and the emulsion. (x 700.)
counts were made of 45 individual A2 females, of known parity, from nine Ax
parents. A2 females of parity I contained 8-27-15-7 x 10~14 moles/female, thus
showing a nearly twofold individual variation. When contrasted with the fourfold individual variation in label content in their Ax parents (see above),.these
data provide further evidence that much of the A± variation is due to surfacebound label which cannot be transmitted. In each of the nine sibships studied,
with few exceptions, A2 females of increasing parity contained decreasing
amounts of label.
(3) For purposes of comparison, a single experiment (Ab) was done with the
closely-related species A. brightwelli (inbred clone 5B4S76; Birky, 1967). This
species produces mictic females in response to vitamin E more readily than does
112
C. W. BIRKY, JR. AND J.J.GILBERT
A. sieboldi, but forms only weakly developed body-wall outgrowths, and those
only after a lag of several generations (Birky (1964) and unpublished; see also
Kiechle & Buchner (1966), where this species is erroneously identified as A.
sieboldi). The transfer and loss patterns for this species appear to be very similar
to those for A. sieboldi. Note especially that in both species, A3 males receive a
smaller portion of their parents' label than do A3 females; this is not due to
smaller amounts of label in their parents, for the amount of label is similar in
mictic and amictic females of the A2 generation.
The loss of label from rotifers to their environment can be estimated by comparing the total label in a group of females plus their offspring with the label
in a sample of the same group of females taken within 24 h after their birth and
before their offspring are born. The results of these calculations are shown in
Tables 3 and 4. In general, an Ax or A2 female loses 40-50 % of her total label
Table 3. a-Tocopherol in Asplanchna: percent loss by Ax females, and percentage
of available Ax label received by A2 females (calculated excluding label lost or
already transmitted)
A2
Exp.
A1
7
9a
9b
11
12HD
12LD
100
100
100
100
100
100
Ab
100
I
II
—
4-4
30-7 42-3
13- 4*
24-1 260
24- 2
25- 1
fl5.5f\
I\ 10-51/
r-il\
III
—
37-2
28-8
29-2
—
.—
_
—
IV
19-7
—
24-7
•
—
_
—
V
VI
A;
Lost
—
26-8
—
—
—
—
_
—
7-8
—
—
—
—
_
77-8
8-3
19-2
15-2
38-1
17-7
18-8
49-2
45-5
49-4
32-5
63-6
39-5
50-3
—
—
* Animals counted after birth of some A3 daughters and loss of some label.
t Amictic females.
J Mictic females.
to the environment in the course of producing four to six female offspring,
which is the usual maximum under our conditions. A loss of more than 60 %
is seen at low population densities (Exp. 12LD, discussed below), in the parity
IV A2 females of Exp. 11 (where the value is uncertain due to low counts in
their A 3 progeny), and in amictic A2 females of A. brightwelli. A large portion of
this loss of label may occur early in life, coincident with the birth of the first
one or two offspring (see Ax in Exp. 12, A2 in Exp. 9b, and Exp. Ab).
Tables 3 and 4 also show the percentage of available label transferred from
mother to offspring; this is calculated excluding both the label which is lost and
the label which has been transferred to offspring already born. A comparison
with Table 2 shows that these percentages are relatively constant for offspring
of different parity, at least through parity V. For the transfer from A2 to A3
Control of development by vitamin E
113
females, the results show an average transfer of about one-third of the available
parental label.
For the purposes of a later discussion, the following generalizations suffice to
describe the results: (1) in each generation, about one-half (rarely two-thirds) of
the total a-tocopherol in a female is lost to the environment; (2) of the remaining
a-tocopherol, nearly all is transferred to offspring: (3) approximately one-third
of the available a-tocopherol in a female at any given time is transferred to her
next female offspring; and (4) each male offspring of a mictic female receives
about one-tenth of its parent's available label.
Table 4. a-Tocopherol in Asplanchna: percentage loss by A2 females and percent
of available A2 label received by A3 females or males (calculated excluding label
lost or already transmitted)
A3
Exp.
7
9b
11
11
Ab
Ab
A2
100
100*
lOOf
100t
10011
1001f
I
11
III
32-6
—
22-9
—
—
39-7 31-5 37-3
45-5§
22-3
—
—
—
—
—
IV
<?
11-4
—
—
—
37-5§ —
—
—
—
6-8
—
A;
Lost
62-6
57-1
9-3
17-8
26-8
47-2
10-5
36-8*
410
67-5
65-7
301
* Based on A 2 from Exp. 9a.
t Parity I.
j Parity IV.
§ Value uncertain because counts very low.
|| Amictic females.
"I Mictic females.
Correlation of vitamin E content with response
In some experiments, the BWO response of the animals to vitamin E was
measured by determining their morphotypic scores. A positive correlation is
apparent between morphotypic scores and a-tocopherol content. This can be
seen in Exp. 9 (Table 2), where the A2 females of increasing parity show decreasing label content and decreasing mean scores. In Exp. 11, the decrease in
morphotypic score with parity in the A2 and A3 generations is less striking, but
it is clear that there is an overall decline in response between generations. In
addition, A 3 females from A2 mothers of parity IV have both a lower response
and a lower label content than those from parity I A 2 mothers. We have applied
the Spearman rank correlation test to these data; the correlation between
a-tocopherol content and mean morph score is highly significant in Exp. 9
(P < 001) and significant in Exp. 11 (001 < P < 005).
Birky (1969) has shown that females raised at higher population densities are
more sensitive to a-tocopherol. This phenomenon was used in Exp. 12 (Tables
8
EMB27
114
C. W. BIRKY, JR. AND J.J.GILBERT
2 and 3) as an additional test for correlations between a-tocopherol content and
the BWO response. Ax females labelled for 18 h at a density of 10 females/ml
(12H) incorporated more a-tocopherol and transferred a greater proportion of
their total label to their first-parity A2 daughters than did females labelled at a
density of 1 female/ml (12L). The more effective transmission was due to a lower
loss of label in the high-density females. This effect may be quite independent of
the soluble sensitizing factor(s) demonstrated by Birky (1969); it is possible, for
example, that label which is lost is promptly re-ingested, more rapidly at higher
population densities. Whatever the mechanism, it is clear that the higher label
content of the high-density A2 females was correlated with higher morphotypic
scores. Taken together, the results of our experiments indicate a clear correlation
between the a-tocopherol content of the rotifers and the degree of their BWO
response.
Table 5. [3H]dl-<x-tocopherol in embryonic and
adult tissues of Asplanchna sieboldi*
Tissue
Ax
A'xt
mature § embryos
postmitoticjl embryos
mitoticij embryos
total embryos
embryos + A^
fluid
(Ax — A^ + embryos)
Label/animal
(dpm)
Moles xlO- 1 5
3650
1920
28.6
11.2
13.0
—
—
657
336
516
201
235
546
391
100
51-1
7.8
3.1
3.6
8.3
—
85-9
13.2
5.1
6.0
14.0
100
—
266
40-5
—
% of Axf
% of Alt
+ embryos
* Ai females labelled 20 h.
t Calculated per animal (adult or embryo).
% A[ = Ai females after removal of embryos and fluid from uterus and pseudocoel.
§ Embryos in the last 5 h of development, with well-developed organs, taking up fluid and
undergoing morphogenetic growth of the body wall.
|| Embryos in ca. 1-\A h of development, characterized by morphogenesis and differentiation of most organ anlage in the absence of mitosis.
^ Embryos in first ca. 6 h of development, during which occurs all mitotic divisions,
considerable cell differentiation, and 'gastrulation'.
Localization of label in embryos and body-cavity fluids
In order to directly demonstrate the presence of a-tocopherol in embryos and
to determine the approximate stage of development at which it is transferred
from parent to daughter, Ax females were labelled for 20 h, washed, and
then drawn into a fine-bore pipet in order to force out their embryos, along with
the maternal uterine fluid and much of the pseudocoel cavity fluid. The embryos
were collected in water, pooled into three groups representing different developmental stages, and counted in Bray's. The remaining maternal tissues were
counted separately. The results are shown in Table 5. All stages of embryos were
Control of development by vitamin E
115
heavily labelled; the 'mature' embryos, which were in the final developmental
stages of swelling due to uptake of fluid and were nearly ready for birth, contained more than twice as much a-tocopherol as earlier stages. About 41 % of
the label initially present in the females could not be accounted for by the sum
of the label in the embryos and in the maternal tissues after the embryos and
fluid were removed. The bulk of this radioactivity is believed to represent cctocopherol in the maternal uterine fluid and/or pseudocoel cavity fluid, which
was lost during removal of the embryos.
Table 6. Thin-layer chromatography of acetone extracts of female
Asplanchna sieboldi
/o of total dpm
Fraction
18
17
16
15
14
13
12
11
10
9
8
7
6
5
4
3
2
1
corrected
%a-toc.||
3
Ax*
Hal
0
0-2
0-5
0-3
01
01
01
01
0-3
0-3
0-5
0-3
0-5 t 0-4
70-8 + 2-8
6-5 62-7
1-5 16-5
1-4
1-3
0-9
0-9
10
50
2-5
0-4
3
O9
A;*
3
HaT
A2*
51
4-4
—
—
0
0
0
0
01
01
0-4
0-6
0
01
0-2
0
0-2
0-3
0-8
0-2
0-9
0-4
0-8
0-5
1-8
76-5| 84-4| 88-8|
2-8
50
2-5
1-2
1-4
0-7
2-6
0-5
0-6
0-5
0-3
0-3
0-41 1-01 0-21
h9\
2-3|
4j2|
0-8
1-7
7-8
1-8
4-6
1-4
QQ 0 /
98%
85% 98% 103%
1« °2-9
H r
2-8
oo/,
—
3
HaT A'*
—
—
0
3.4
0
0
01
0
0-3
1-9
1-7
0-6
0-2
0-8
4-3
21
63-5| 49-6|
1-5
20
2-7
2-3
1-4
0
11
0-5
6-11 7-7|
*2|
4-5|
3-8
8-3
12-4 130
98%
77%
3
HaT
A3t
—
—
0-2
0
1-2
1-3
2-9
6-8
1-2
1-7
1-5
3-2
0-9
1-6
0-9 \ 1-7
14-8 i
47-5 55-3|
3-4
5-1
11
0
1-1
0
1-5
0
1-8
1-7
16-5| 11 -2|
3-8 10-5
98% 87%
* A l5 A^, A2, and A'2 from one experiment, in which the A1 were labelled 20 h in 10~6 M
[ 3 H]i//-a-tocopherol; the A2 females, of parity I, had produced a mean of two offspring/female
before being counted, and were inadvertently allowed to starve for several hours shortly
after birth.
f A3 females were parity I—II; their parity I—IIA2 mothers were born to A : females labelled
19 h in 4-5 x 10~ 7 M [3H]<//-a-tocopherol.
t Location of a-tocopherol.
§ Location of a-tocopheryl quinone.
|| Calculated assuming [ 3 H]a-tocopherol was 98 % pure.
Autoradiographic localization of labelled a-tocopherol
Additional evidence for a localization of a-tocopherol in the maternal uterine
fluid was obtained by autoradiography. Most autoradiographs of Ax and A2
females showed no obvious localization of label in any tissues except for the
8-2
116
C. W. BIRKY, JR. AND J.J.GILBERT
expected light concentration in stomach cells; embryos were also weakly labelled.
Ax females in two blocks, however, differed from others in showing a dense,
osmium-stainable material in the uterus. This material showed a marked
accumulation of label (Fig. 2). We believe that the dense material represents the
uterine fluid or some component thereof, in which labelled a-tocopherol is
concentrated. Probably, for unknown reasons, the material was not well fixed in
most blocks and, together with the label, was extracted during dehydration and/
or embedding.
Identification of labelled material by thin-layer chromatography
The results of the most successful thin-layer chromatographs of extracts of
labelled females are summarized in Table 6. Examination of the data for control
runs of highly (advertised as 97-98 %) pure [H3]a-tocopherol indicates that some
oxidation of the tocopherol is occurring on the plate, in spite of the precautions
described under Materials and Methods. It is therefore necessary to correct the
percentage of a-tocopherol found in the extracts for this oxidation. When this
is done, it is found that between 76 and 100 % of the labelled material present
in Al5 A2 and A 3 females migrates as a-tocopherol. This material is therefore
probably undegraded a-tocopherol. Variable amounts of what is probably
a-tocopheryl quinone or perhaps tocopheronolactone were also found in all
runs (authentic samples of these two compounds have similar Rf values under
our conditions). No marked accumulations of radioactivity were found with
Rf values of dimeric oxidation products (the spiro dimer and 5,5'-Bi-a-tocopherol, structures I and III respectively of Nelan & Robeson, 1962, and 5,5'methylene bis-(y-tocopherol)).
DISCUSSION
Four conclusions emerge directly from our experimental data.
(1) Of the a-tocopherol consumed by female Asplanchna, about half is lost to
the environment in each generation; the remainder is largely present in apparently
undegraded condition. This stability of a-tocopherol in vivo is in marked contrast
to its extreme susceptibility to oxidation in vitro; it indicates the existence of a
mechanism for the protection of the molecule from metabolism (including
extensive use as an antioxidant) and its preservation for other purposes. The
fate of the lost material is unknown. It may be metabolized before it is lost, but
if so, the retention time of derivatives of the labelled methyl group, at least,
must be quite short. Nor do we know the mechanism of loss. Since a large part
of the labelled a-tocopherol seems to be localized in a uterine fluid, it is possible
that it leaks out during birth; this would explain why much of the loss occurs at
about the time of birth of the first offspring.
(2) The remaining a-tocopherol present in each generation is almost entirely
transmitted to offspring. In the Ax generation, this means that vitamin E must be
efficiently transferred from the stomach to the pseudocoel cavity fluid and hence
to the embryo via the uterus and possibly also via the vitellarium. The maternal
Control of development by vitamin E
111
vitellarium, which functions analogously to nurse cells in other organisms,
cannot be the only source of vitamin E for embryos, since vitamin E fed to
mature females can modify the development of embryos already detached from
the vitellarium (Gilbert, 1968; Birky, 1968). This is confirmed by the autoradiographic localization of labelled a-tocopherol in the uterine cavity, and by
the scintillation counting experiment which indicated that at least 40 % of the
total label in Ax females is in the pseudocoel or uterine fluids, or both.
Krishnamurthy & Bieri (1963) have studied the stability and retention of
^/-a-tocopherol-5-methyl-14C, administered orally to rats and chicks partially
depleted of vitamin E. In rats, about one-third of the total dose was excreted or
could not be recovered after 24 h; about 1 % was excreted each day for the next
20 days. The bulk of the excreted and retained label was in the form of octocopherol. Retention was somewhat lower in the chicks, and a considerable
amount of the a-tocopherol was degraded after 24 h. No attempt was made to
detect radioactivity in the offspring.
By extrapolation from our autoradiographs, it seems likely that vitamin E in
Asplanchna would also be localized in body fluids in the A2 and subsequent
generations, even though these animals have obtained their vitamin E from
their parents rather than by ingestion. This has been verified by Oliver (1970),
who has obtained the combined pseudocoel and uterine fluids by dissecting
females under oil, and shown that this fluid can induce the BWO response in the
progeny of females reared from birth in the fluid. The response was obtained
with the fluid from females whose parents had been fed the water-soluble
derivative, a-tocopheryl polyethylene glycol 1000 succinate (TPGS), and also
from control females taken from cultures with no added vitamin E. The fluids
were also fractionated by thin-layer chromatography and the fractions tested
separately; the major inducing component in the fluid from induced females
migrated with the Rfof a-tocopheryl succinate (presumably derived from TPGS),
while the major inducer in the fluid control females was identified by its Rf as
a-tocopherol - presumably obtained from the traces identified in Scottish
grass infusion by Gilbert & Thompson (1968), and responsible for the weak
BWO responses often observed in control cultures. In addition, Oliver (1970)
has shown that embryos taken from control females can be induced to form
weakly developed BWO's by rearing them in vitro in the combined pseudocoel
and uterine fluids taken from induced females.
(3) Successive offspring receive smaller amounts of their parental a-tocopherol,
but approximately constant amounts of that remaining after the birth of their older
sibs; males receive less than females. In rotifers, one oocyte is matured at a time.
After maturation, which requires about 6 h, the oocyte is set free from the
vitellarium and undergoes complete development in the uterus (about 20 h)
while the next oocyte begins maturation. Mature amictic females thus contain
four to five female offspring in different stages of development; mictic females
produce more (male) offspring and generally carry more embryos at one time.
118
C. W. BIRKY, JR. AND J.J.GILBERT
These observations suggest a possible model for the transmission of vitamin E
from generation to generation. We suppose that vitamin E is specifically localized in the uterine fluid, and that most of this localization occurs before reproductive maturity. The molecules are absorbed by maturing embryos from the
uterine fluid; as successive embryos mature and are born, the concentration of
vitamin E and consequently the amount available for absorption declines. Male
embryos, competing for vitamin E with a larger number of siblings in utero,
would obtain smaller amounts of label than females.
(4) There is a positive correlation between the a-tocopherol content of females
and their BWO response. This generalization cannot, of course, apply to Ax
females, which cannot respond because they were not exposed to the a-tocopherol
as embryos. Other possible exceptions will be considered below.
Our experiments were begun in order to provide at least an indirect test of
two hypotheses about the role of vitamin E in developmental polymorphism.
The first of these hypotheses is that vitamin E itself acts directly upon embryos
in utero to control development, i.e. that it is the intrinsic as well as the extrinsic
inducer. Alternative hypotheses would be that the intrinsic inducer is a metabolic
derivative of a-tocopherol, or another molecular species or a metabolic state
induced in the females by a-tocopherol. If a-tocopherol is the intrinsic inducer,
it would have to be transmitted intact in reasonable quantity from a pulse-fed
A t female to her progeny through as many as four generations in order to
explain the short-term inheritance of the BWO response. It might also be
possible to show at least a rough correspondence between endogenous atocopherol levels and the magnitude of the BWO response. Both of these
conditions have been met in the experiments reported here. While this does not
directly prove that a-tocopherol is an intrinsic inducer, it argues strongly for the
hypothesis.
In this connexion, two observations of Birky & Power (1969) posed some
difficulty for the identification of a-tocopherol as the intrinsic inducer, and require
special comment. In many experiments where Ax females were fed vitamin E
during their first day of life, it was found that (1) their first three or four A2
progeny showed progressively increasing morphotypic scores, whereas our
present data indicate that they should have contained decreasing amounts of
a-tocopherol, and (2) mean morphotypic scores remained constant or rose
between the A2 and A3 generations, and then declined approximately linearly,
while a-tocopherol content should decline logarithmically in every generation.
Our data suggest two probable explanations. First, it is possible that atocopherol in Ax females is localized in the uterine fluid rather slowly, so that
parity IA 2 embryos absorb most of their a-tocopherol too late in development to
influence morphogenesis (cf. Table 5). Thus, although their content of atocopherol would be greater than that of embryos of later parity and of the
next (A3) generation, their response would be lower.
The second explanation is suggested by the data of Gilbert & Birky (1971),
Control of development by vitamin E
119
who determined the BWO response in A2 females when their A± parents were
fed concentrations of a-tocopherol ranging from 10~ n M to 5 x 10~7 M. Their
data indicate a maximal response at about 5x 10~ 8 M; the response declines
steeply below this, while higher concentrations may actually be slightly inhibitory. The experimental conditions used by Gilbert & Birky (1971) are not
strictly comparable to those used in the present study. However, we have used
our present data on uptake and transmission to estimate the actual a-tocopherol
content of the A2 females in the previous study, and to plot a rough curve of
concentration against response. The results show a peak response at 3 x 10~14
moles/female, with higher contents being inhibitory. From an examination of
the data in Table 2, it seems likely that the first-born A2 females of Birky &
Power (1969) actually contained inhibitory amounts, and the A3 and later A2
females contained optimal amounts, of a-tocopherol. Our calculations from the
data of Gilbert & Birky (1971) also indicate that their A2 females showed a
positive BWO response when they contained about 5 x 10~16 moles of atocopherol, and no response at about 1 x 10~16 moles. This estimate of the
minimum amount of a-tocopherol required to induce a BWO response in A2
females agrees well with the data in the current paper. In Exp. 11 (Table 2), A3
females showed a positive response at about 9 x 10~16 moles and no response at
5 x 10~16 moles. In Exp. 7, we can estimate that the A4 and A5 females contained
about 9 x 10~16 and 3 x 10~16 moles/female respectively (by extrapolation from
the A2 and A3 data in Table 2); the A4 females showed a significant BWO
response, while the A5 did not. Our data thus suggest that, in any generation,
about 5-10 x 10~16 moles, or 3-6 x 108 molecules, of a-tocopherol are required to
induce a recognizable BWO response. This, of course, is in addition to the basal
content of a-tocopherol found in all females and obtained from the Scottish
grass infusion.
Our second hypothesis about vitamin E concerns the adaptive significance of
the use of this molecule as a necessary, if not sufficient, stimulus for the production of mictic females. We have suggested (Gilbert & Thompson, 1968;
Birky, 1969; and especially Gilbert, 1971) that high levels of vitamin E may be
essential for the fertility of male, but not female, Asplanchna. If so, it would be
entirely reasonable for rotifer populations to have evolved mechanisms which
would require the availability of high levels of vitamin E before sexual reproduction is initiated. Since male rotifers do not feed, they can only obtain
vitamin Efrom their parents; if it is required for fertility, a mechanism would
also have to be evolved to ensure that dietary vitamin E is protected from degradation and efficiently transmitted from parent to offspring. Our present data
show that this is indeed the case. It seems to us extremely unlikely that the
remarkable behavior of vitamin E, which sets it apart from the bulk of dietary
macromolecules, is fortuitous; it probably indicates a special role for the
molecule. Our hypothesis was originally suggested by the necessity of vitamin
E for male, but not female, fertility in certain other organisms, and is consistent
120
C. W. BIRKY, JR. AND J. J. GILBERT
with the autoradiographic localization of a-tocopherol in the male testis (Gilbert,
1971).
We thank Dr George Malacinski for helpful comments on the manuscript and Mrs Maxine
Bean for expert and enthusiastic assistance. The work reported in this paper was supported by
National Science Foundation Research Grant GB-7717 to J. J. G. and U.S. Public Health
Service Research Fellowship 1 F03 GM 43071-01 to C. W. B., Jr.
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{Manuscript received 12 June 1971)