/. Embryol. exp. Morph. 94, 221-230 (1986)
221
Printed in Great Britain © The Company of Biologists Limited 1986
Analysis of the cytosolic proteins of chick embryo
gonads by two-dimensional gel electrophoresis
J. S A M S E L 1 , B. L O R B E R 2 , A. P E T I T 1 AND J.-P. W E N I G E R 1
l
Laboratoire de Zoologie et d'Embryologie experimentale de V University Louis
Pasteur, 12 rue de VUniversite, F-67000 Strasbourg, France
2
Laboratoire de Biochimie de Vlnstitut de Biologie MoUculaire et Cellulaire du
CNRS, Strasbourg, France
SUMMARY
Gonads and mesonephros dissected from normal chick embryos, as well as a blood sample,
were labelled in vitro with [35S]methionine and [14C]leucine. The patterns of cytosolic protein
synthesis of the different tissues were analysed using two-dimensional (2-D) polyacrylamide gel
electrophoresis. Three developmental stages, i.e. 8, 12 and 17 days of incubation, were
investigated.
Five sex-specific proteins were detected in the male and two in the female. In the testis, only
one protein is already present at the 8-day stage. In the ovary, one protein exists since the 8-day
stage, but it is also synthesized in the mesonephros. The second ovary-specific protein appeared
only at the 12-day stage.
INTRODUCTION
The mechanism of gonadal sex differentiation, in vertebrates as well as in
invertebrates, is still unknown. For some years, the H-Y antigen has been thought
to be the testis-determining factor in mammals, but this hypothesis is now being
abandoned (McLaren etal. 1984).
In the present study using two-dimensional gel electrophoresis, we wanted to
see whether testis- or ovary-specific proteins could be detected in the chick embryo
at different stages, our final goal being the isolation and characterization of one or
several proteins responsible for testicular or ovarian differentiation.
Three developmental stages, i.e. 8, 12 and 17 days of incubation, were investigated. Although at all three stages the gonads were completely separated
from the mesonephros, mesonephric samples have also been analysed in order to
detect any possible contamination of the gonadic samples with mesonephric
remnants. In the same way, since we observed that the blood supply of the ovary
was of much greater importance than that of the testis, a blood sample was
included in the study.
As regards related studies, the work of Miiller etal. (1984) should be mentioned.
These authors have shown that gonad-specific proteins exist in the rat foetus.
Key words: chick embryo gonads, 2-D electrophoresis, sex-specific proteins.
222
J. SAMSEL, B. LORBER, A. PETIT & J.-P.
WENIGER
MATERIALS AND METHODS
Animal material
8-, 12- and 17-day-old normal chick embryos of the White Leghorn strain were used. Both
testes, the left ovary and mesonephric tissue from males and females were taken for analysis at
each stage. A blood sample was drawn with a heparinized syringe from the omphalo-mesenteric
vein of a 17-day-old embryo.
Organ culture and radiolabelling
Organs were dissected under sterile conditions in Tyrode solution, cut into small fragments
and pooled to obtain about 10 mg of tissue per culture dish containing 0-125 ml of Tissue
Culture Medium 199 (Laboratoires Eurobio, Paris, France) and lOOjuCi of L-[35S]methionine
(HOOCimmol"1, New England Nuclear) and lOOjuCi of L-[14C]leucine pOOmCimmor1,
Commissariat a l'Energie Atomique, France). The number of organs required to obtain 10 mg of
tissue in successive stages varied. For instance, five ovaries or pairs of testes of 17-day-old
embryos were needed.
After culturing the organs at 37-5°C for 2h, they were rinsed four times (1x0-2ml and
3x0-5 ml) with ice-cold Tyrode solution to remove free radioactive aminoacids and secreted
proteins. Tyrode solution contained O-lmM-phenylmethylsulphonyl fluoride in order to avoid
proteolysis. 100/il blood samples were diluted with 0-125 ml of culture medium containing
radioactive aminoacids as mentioned and were also incubated for 2h.
Preparation of samples for electrophoresis
(1) Cytosol samples
Cultured organs were suspended in 0-5 ml of Tyrode solution containing the protease
inhibitor. They were homogenized by 15-20 strokes in a glass-Teflon homogenizer. The
homogenate was centrifuged (4°C) successively at 480 g for 20min, 13 500 g for 1 h and 105 000 g
for 4h. The last centrifugation separates the ribosomes, and the supernatant constitutes the
cytosolic fraction, which was lyophilized.
(2) Culture medium samples
At the end of the 2 h culture period, each culture medium was harvested and added to the first
0-2 ml of rinsing solution. Each sample was then centrifuged (4°C) at 13 500g for l h and
105 000g for 4h. The last supernatant represents the culture medium, which was lyophilized.
(3) Blood sample
After the 2 h incubation period the content of the culture dish was collected and homogenized
as in (1). The sample was then centrifuged (4°C) two times as in (2); the supernatant represents
the blood sample, which was lyophilized.
Two-dimensional gel electrophoresis
Two-dimensional electrophoresis analysis was performed according to the procedure of
O'Farrell (1975). Each of the lyophilized samples was dissolved in 50jul of lysis buffer. 2 (A
of the protein solution were withdrawn to determine the amount of radioactivity incorporated
into TCA-precipitable material. 40^1 aliquots containing identical amounts of incorporated
radioactivity were prepared by diluting the samples with lysis buffer. The 40 jul aliquots of cytosol
samples contained lxl0 6 ctsmin~ 1 and approximately 80jUg of protein. In the culture medium
samples, the TCA-precipitable material was so small that the 40jul aliquots contained only
lxK^ctsmin" 1 . The blood sample contained lxK^ctsmin" 1 . The different aliquots were then
stored at -80°C before they were layered onto cylindrical isoelectric focusing gels (3x120 mm)
prepared and prerun as described by O'Farrell (1975).
After electrofocusing at 20°C, the gels were equilibrated and shaken in sodium dodecyl
sulphate buffer for 40min at room temperature. The pH gradient was determined immediately
Protein synthesis in chick embryo gonads
223
after the migration by slicing a control gel into 5 mm segments and extracting each slice with 2 ml
of double glass-distilled water for l h prior to measuring the pH at 20°C. Slab gels (160 x
140x1 mm) containing 10-5 % (w/v) polyacrylamide and 0-1 % (w/v) SDS were used for the
second dimension. The apparent molecular weight of the polypeptide chains was estimated by
comparison with reference proteins (Pharmacia, Low Molecular Weight Calibration Kit) run
simultaneously. Proteins were fixed in the gel by immersion in a 10 % (v/v) trichloroacetic acid
solution for 30min.
Protein staining and autoradiography
Slab gels containing both radiolabelled proteins of samples and non-radioactive reference
proteins were first stained by the classical technique using PAGE Blue 83 (BDH chemicals,
Poole, England) at 0-1% (w/v) in methanol/water/acetic acid 25:65:10, and photographed.
After this step, the gels were dried on Whatman 3MM chromatographic paper and subsequently
exposed to FUJI RX2 film for autoradiography (16 days for lxl0 6 ctsmin~ 1 ). Prolonged
exposure time was preferred to the treatment with an autoradiographic enhancer to obtain a
greater sharpness of the spots. Completely reproducible patterns were obtained either from two
gels of the same sample or from different radiolabellings of the same tissue.
RESULTS
High resolution two-dimensional gel electrophoresis was used to compare the
cytosolic proteins synthesized during a 2h labelling period by normal male and
female chick embryo gonads at various developmental stages. Comparisons with
mesonephric and blood samples were also performed.
A total of about 600 protein spots were compared for each tissue examined. The
clearest resolution was found at molecular weights (Mr) above 30xl0 3 . Beside the
quantitatively major proteins common to all tissues, 64 protein spots were
accorded particular attention. These proteins undergo reproducible stage-specific
changes and some were found to be also sex-specific. The results are summarized
in Table 1.
(A) Sex-specific gonadal proteins
A thorough comparative analysis of gonadal protein patterns of 8-, 12- and 17day-old normal chick embryos (Figs 1-3) revealed the presence of sex-specific
proteins in both male and female gonads. Proteins 1-15-16-24 and 25 are testisspecific and proteins 33 and 47 are ovary-specific. The approximate molecular
weights and isoelectric points of these proteins are given in Table 2. Proteins 1-1624-25 and 33 are not synthesized at the 8-day stage, and protein 1 appeared only at
the 12-day stage. So, proteins 15 and 47 constitute the earliest differences between
testicular and ovarian tissues.
(B) Proteins common to testis and ovary
Except for the sex-specific gonadal proteins, the male and the female gonadal
protein patterns are very similar. Most proteins are present at all stages. Only
a small number are not synthesized at 8 days. The quantitative variations of the
protein spots 1-50 in relation to sex and developmental stage have already been
listed (Table 1). Table 3 gives a summary of the quantitative variations between
±
+
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±
±
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7
± , at the limit of detection; + + + , very abundant.
26
+
4
7
4 8 +
49
±
50
stages: 35
+
+
±
46
+
+
++
±
42
+
36
±
+
+
+
±
40
j-j.
+
±
43
j-j-j.
Stage 12 Stage 17 Stage 8 Stage 12 Stage 17
+
+
±
+
+
+ +
+
+
+
++
++
+
+
+
+
±
±
±
+
+
±
+
+
++
++
++
++
±
+
±
±
±
+
±
±
±
±
+
++
±
±
++
+
+
+
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+
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+
±
±
+
+ + + +
+
+ + + +
+
+
±
±
±
++
± ±
++
+
+
±
++
++
±
++
++
+
+
±
+
+
Stage 8
±
2 8 +
29
3
0
±
±
31
±
+
32
33
34
+
3
5
±
++
36
±
±
37
+
38
+
39
+
4 0 ±
41
+
+
42
±
43
+
4
4
±
±
45
±
±
4 6 ±
2
+
±
Stage 12 Stage 17
2 3 +
+
+
±
+
±
24
±
±
25
++
Number of numbered protein spots present at the different developmental
+
22
±
±
±
+
+
+
±
±
+
+
+
+
++
+
+
Stage 8
Stage 8 Stage 12 Stage 17
~1
+
2
++
3 + + + +
+
4
++
++
5 ±
6 +
±
±
7
++
+
++
8
+
+
+
+
+
9
+
+
+
+
+
1 0 +
+
±
11
++
++
++
12
+
±
13
+
+
+
+
+
14
+
+
+
15
±
±
+
16
±
±
17+
+
+
18
+
+
+
19
+
+
20
±
+
+
21
±
+
+
Table 1. Estimation of the relative abundance of proteins 1-50 in the cytosol of male and female gonadsfrom 8-, 12- andl7-dayold normal chick embryos
Male
Female
Male
Female
+g
*
z
y
^
w
>
^
"
q
g
w
g
r
w
^
^
>
I
«
~
to
to
Protein synthesis in chick embryo gonads
225
the 8- and 12-day stages and the 12- and 17-day stages for the 50 proteins analysed.
Important qualitative variations in the synthesized proteins were detected in both
male and female gonads between 8 and 12 days. Fourteen protein spots with an
elongated shape (named a-n in Fig. 1) are only present in 8-day-old organs.
Fourteen other protein spots (named A-N) with approximately the same molecular weights as protein spots a-n, but with different isoelectric points, appeared at
12 days (Fig. 2) and were also present at 17 days (Fig. 3).
(C) Comparison of the gonadal protein patterns with those of the gonadal culture
medium, mesonephros and blood
Protein patterns of culture media of both male and female gonads comprised
only a few spots. None of these spots are specific to one sex. By comparison with
the gonadal cytosolic protein patterns no superimposable spots could be seen.
On the other hand, protein patterns of mesonephros taken from male and
female and blood protein pattern showed numerous spots. The mesonephric and
the gonadal cytosolic protein patterns are almost identical. Among the 64 gonadal
protein spots subjected to a thorough comparison, 53 are present in the
mesonephros in variable quantities and only the. sex-specific proteins, except
Table 2. Apparent molecular weights and isoelectric points of the sex-specific proteins
Apparent molecular weight (MT)
(x 10~3)
Protein number of proteins subi
1
180
15
36
16
36
24
22
25
15
33
55
47
27
Isoelectric point under
denaturing conditions in
the presence of 8-5 M-urea
6-4
6-5
6-2
6-6
5-7
4-3
4-5
Table 3. Quantitative variations of protein spots 1 to 50 during the development of
testis and ovary
A
B
cf
$
cf
$
Rates (in %)
- more abundant proteins
- less abundant proteins
- proteins without quantitative variation
Total
40
25
19
56
24
17
59
13
7
80
100%
100%
100%
100%
49
11
Recall that in both sexes, not all the 50 numbered protein spots are present at all the different
development stages. Number of protein spots present at 8,12 and 17 days are the following: 35,
46 and 42 in the testis and 36, 40 and 43 in the ovary.
(A) Comparison between organs of 8- and 12-day-old embryos; (B) comparison between
organs of 12- and 17-day-old embryos.
226
J. SAMSEL, B. LORBER, A. PETIT & J.-P.
WENIGER
pH
<f
1CT3
94
26
67
43
30
20-1
94
67
43
30
20-1
Fig. 1. Two-dimensional gel electrophoretic patterns of the cytosolic proteins of the
gonads from normal male (cf) and female ($) 8-day-old chick embryos. The most
relevant spots have been numbered from 1 to 50. Arrows point to a spot or indicate the
lack of a given spot. Circled numbers indicate the sex-specific proteins. Arrows with
small letters indicate protein spots present at 8 days only.
227
Protein synthesis in chick embryo gonads
pH
Mr x
10" 3
94
67
43
-
i
/ •
30
r.f f-
20-1
•
^
Ifllrf
94
20-1
Fig. 2. Two-dimensional gel electrophoretic patterns of the cytosolic proteins of the
gonads from normal male (cT) and female ($) 12-day-old chick embryos. Capital
letters indicate protein spots present at 12 and 17 days. These proteins have approximately the same apparent molecular weights as those designated by small letters
at 8 days (Fig. 1), but they have different isoelectric points. Other informations about
these gels is the same as that given in the legend to Fig. 1.
228
J. S A M S E L , B . L O R B E R , A . P E T I T & J . - P .
7
WENIGER
pH
4
10"3
B
94
26
35
67
43
30
20-1
94
67
43
30
20-1
Fig. 3. Two-dimensional gel electrophoretic patterns of the cytosolic proteins of the
gonads from normal male (cf) and female ($) 17-day-old chick embryos. Information
about these gels is the same as that given in the legends to Figs 1 and 2.
Protein synthesis in chick embryo gonads
5
PH
4
229
Mrx10"3
43
30
20-1
Fig. 4. Cut of two-dimensional gel electrophoretic pattern of the cytosolic proteins of
the mesonephros from normal male 8-day-old chick embryo. Information about this
gel is the same as that given in the legend to Fig. 1.
protein 47, are absent. A large spot covers the area of protein 47 (Fig. 4), which,
though sex-specific, is therefore not ovary-specific. None of the seven sex-specific
proteins were detected in blood.
DISCUSSION
The present study is a comparison of the cytosolic protein patterns of the gonads
from normal male and female 8-, 12- and 17-day-old chick embryos. The protein
patterns were analysed by two-dimensional polyacrylamide gel electrophoresis
followed by autoradiography. Preliminary experiments with 17-day-old gonads
cultured for 10 min, 30 min, 2 h or 24 h had shown that there was no increase in the
number of radioactive spots after 2h of incubation. So, a 2h culture period was
currently used. Radiolabelling with [35S]methionine and [14C]leucine was performed simultaneously in order to increase the sensitivity of the method, which
separated proteins of isoelectric points between pH 4 and 7 and apparent molecular weights (A/r) of 14 to 20xl0 3 for the subunits.
By this method we demonstrate for the first time the existence of gonad-specific
proteins in the chick embryo. Two of these proteins, namely protein 15 in the testis
and protein 47 in the ovary are already present at the 8-day stage, i.e. shortly after
the stage of morphological sex differentiation, which coincides with the 6th day of
incubation. With the exception of protein 47, gonad-specific proteins are only
present in low amounts in the cytosol. Control experiments showed that these
proteins are not secreted into the culture medium.
In spite of our observation that at all stages the blood supply of the ovary was of
greater importance than that of the testis, the absence of the seven sex-specific
proteins in the blood ascertained that these proteins are well formed in gonadal
tissue.
230
J. SAMSEL, B. LORBER, A. PETIT & J.-P.
WENIGER
On the other hand, at all stages, gel electrophoresis of the cytosol from the
mesonephros showed a large spot in the area of ovary-specific spot 47. Short
exposure of these gels confirmed that this spot exactly overlapped spot 47. So, the
ovary and the mesonephros have in common a protein which is absent from the
testis.
It is known from previous work in the field (Carlon, Pizant & Stahl, 1983;
Wartenberg, 1983) that cells of mesonephric origin participate in the formation of
the ovary. Therefore it would not be surprising that both organs synthesize a
common protein. Nevertheless, this would be in contradiction with the results of
other authors who deny that mesonephric cells play a part in the formation of the
ovary (Calame, 1961; Popova & Scheib, 1981; Merchant-Larios, Popova & ReyssBrion, 1984).
The earliest stage investigated was 8 days. It would be interesting to examine
still earlier stages, possibly before morphological sex differentiation and to include
membrane proteins.
In conclusion, it would seem that analysis of the gonadal protein pattern at
various developmental stages constitutes a valuable technique for investigating
gonadal sex differentiation in the chick embryo.
J. Samsel and J.-P. Weniger express their deepest thanks to Professor J.-P. Ebel, Head of the
Institute de Biologie Mol6culaire et Cellulaire du CNRS for hospitality in his laboratory.
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(Accepted 10 December 1985)