- Email: [email protected]
Sertoli cells are the site of interleukin-1α synthesis in rat testis
Molecular ad Cdular Endocrinology, 82 (1991) R13-R16 0 1991 Elsevier Scientific Publishers Ireland. Ltd. 0303-7207/Y
MOLCEL
R13 l/$03.50
02665
Rapid Paper
Sertoli cells are the site of interleukin-lcu Nadine, G&rard
synthesis in rat testis
‘, Viqar Syed I-2, Wayne Bardin 2, NoElle Genetet and Bernard
’
Jkgou ’
’ G. E.R.M., INSERM CJF 91-04, Unilvmitr: de Rennrs I, 35042 Rmne.s, France, ’ The Population Council, New York, NY 10021, U.S.A., und .’ G. U. R. I. F.A., 35036 Rennes, France (Received
Key words: Interleukin-1;
Sertoli
11 September
cell: Spermatogenesis;
199 1; accepted
Paracrine
factor;
16 September
199 1)
(Rat testis)
Summary To elucidate the cellular origin of interleukin-1 (IL-11 in the mammalian testis, we assayed IL-1 activity in culture media of Sertoli cells collected from rats at 20, 35 and 45 days of age as well as in culture media of interstitial, peritubular and germ cells from adult rats. IL-l was detected in Sertoli cell culture media isolated from 35 and 45-day-old rats. At 45 days approximatly twice as much IL-l was produced than at 35 days. In contrast, IL-1 activity was not detected in 20-day-old rat Sertoli cell culture media nor in interstitial, peritubular and germ cell culture media. The Sertoli cell IL-1 activity was specifically neutralized by an IL-la antiserum. It is concluded that Sertoli cells produce IL-la and that this production increases during sexual maturation.
Introduction The mammalian seminiferous tubules are comprised of the somatic Sertoli cells and germ cells whose proliferation is under the hormonal control of LH and FSH as well as of an extremely complex set of paracrine factors (Sharpe, 1986; Jtgou et al., 1988). One of them, an interleukinla-like factor, was identified in the testis of the rat after 30 days of age (Khan et al., 1987; Syed et al., 1988) and in testicular extracts of the adult human (Khan et al., 1988). Using indirect approaches, Khan et al. (1987) and Syed et al. (1988) have suggested that Sertoli cells may be
Address for correspondence: SERM CJF 91-04, UniversitC Beaulieu, 35042 Rennes Cedex,
B. JCgou. de Rennes France.
G.E.R.M., I, Campus
INde
the source of the testicular interleukin-la (IL-la) production, but to date there is no direct evidence to substantiate this hypothesis. In the present study we demonstrate that rat Sertoli cells in vitro specifically secrete IL-la and that the production of IL-la increases with the stages of sexual development. Materials
and methods
Sertoli cell isolation and culture. Sertoli cells were prepared from 20-, 35- and 45-day-old Sprague-Dawley rats according to methods described elsewhere (Mather and Phillips, 1984; Toebosch et al., 1989) and cultured at 32°C in a humidified atmosphere of 5% CO,-95% air in Ham’s F12/DMEM (v/v; Gibco BRL) supplemented with insulin (10 pg/mll, transferrin (5 pg/ml) and gentamicin (4 pg/ml). After 2-3
RI4
Germ cell isolation and culture. Germ cells prepared from adult Sprague-Dawley rat testes by trypsinisation (Meistrich et al., 1981) were submitted to a centrifugal elutriation. The two fractions obtained: pachytene spermatocytes and round spermatids (90% enrichment) were incubated in Ham’s FIZ/DMEM (v/v) enriched with lactate (6 mM), pyruvate (2 mM) and gentamicin (4 ~g/ml). After 24 h of culture the media were kept at -80°C. Interstitial and peritubular cell isolation and culture. Interstitial and peritubular cells were isolated from 20- and from W-day-old SpragueDawley rat testes by collagenase digestion according to methods previously described (Purvis et al.,
days of culture, Sertoli cell monolayers were exposed to a Tris-HCI buffer solution (20 mM, pH 7.4) to remove contaminating germ cells (Galdieri ct al., 1981; Le Magueresse and Jegou, 1988). The day after, cells were incubated for an additional 24 h in medium where transferrin was omitted. The media were kept at -8c1”C and the cells were scraped and frozen at -80 “C until DNA content assay (Valotaire and Duval, 1969). At the termination of the culture, the purity of the Sertoli cell cultures was determined: perituhular cell and germ cell contaminations were less than 7% and 2% respectively for 20-day-old Sertoli cells, 3% and 7% respectively for Sertoli cells from older animals.
/.:
20
35
45
AGE
-I-rl
C PHA
32 16
8
4
2
16
8
4
2
16
20 Days L-IL-la!
curves of IL-I
C: control cells without PHA.
2
35 oays
DILUTIONS
activity obtained with human recombinant Inset: IL-la
4
-J RECIPROCAL
Fig. 1. Dose-response
8
IL-la
and SCCM
from 20-. 35 and 45day-old-rats.
secretion expressed in units per gg of Sertoli cell DNA
at the different
ages.
R15
1978; Mather and Phillips, 1984). The cells were washed to remove most of the contaminating cells after 1 h of preincubation. They were cultured for 24 h and the media kept at - 80 o C. Bioassay of IL-I. IL-l was measured in the different culture media using the murine thymocyte proliferation assay (Scala and Oppenheim, 19831, with minor modifications. Briefly, thymocytes (15 x 10’ ceils/ml) from 4-&week-old CH,/HE female mice were incubated in the presence of phytohemaglutinin (PHA, 20 pg/ml), with various doses of human recombinant IL-la (Boehringer-Mannheim; starting dilution: 50 U/ml> or test material, for 48 h at 37°C. During the last 2 h of incubation [‘Hlthymidine was added. Incorporated radioactivity was determined by liquid scintillation counting and results were expressed as mean counts per min (cpm> of triplicate cultures. In our assay conditions, one unit of IL-1 activity was arbitrarily defined as the ability of test material to double [~H]thymidine incorporation into thymocytes, when compared with PHA stimulated cultures (Syed et al., 1988).
El
scM
m
scLY4 + ANTI IL-la
m
SCCM + ANTI IL-l@
Bioassay of IL-2. IL-2 activity was assayed using a specific bioassay based on the measurement of the proliferation of a cloned murine cytotoxic T lymphocyte line (CTLL 2) (Gillis et al., 1978). Results As shown in Fig. 1, Sertoli cell culture media (SCCM) obtained from 35- and 45-day-old-rats induced a significant dose-dependent proliferation of murine thymocytes, whereas no IL-l activity was detected in the culture media of Sertoli cells prepared from ZO-day-old animals. Germ cell conditioned media as well as interstitial and peritubular cell culture media lacked IL-l activity (data not shown). It is noteworthy that SCCM prepared with cells collected from pubertal rats f45-day-old) displayed approximatively twice as much bioactivity as SCCM prepared with cells collected from prepubertal animals (35-day-old). Of note also is the fact that while the specific IL-la antiserum could block the bioactivity of the
7
.L. . . . . . . . . . .
. . . . .
. . . . .
-! I)... . .
. . ..*
.
.
.
.
.
. .
. .
. .
. .
.
.
.
.
.
.
.
.
a... . . . . . . . . .*..
. . . .
. . . .
..,.
_
45 Days PHA
Fig. 2. Effect of sheep anti-murine
IL-la
1
SCCti
1
and IL-l@ antisera on the bioactivity of IL-I. SCCM were incubated h at room temperature before being assayed.
with the antibodies
1
SCCM prepared from the older Sertoli cell donors, the specific IL-ID antiserum had no effect (Fig. 2). Moreover no IL-2 bioactivity could be detected in any of the media tested (data not shown).
and IL-l@ antisera. This work was supported by I’INSERM (Paris, No. 900406), la Fondation de France (Paris) and NIH grant HD 13541.
References Discussion
Our data confirm previous results showing that peritubular and interstitial cells do not secrete IL-1 Khan et al., 1987; Syed et al., 1988). Therefore, the data reported here demonstrate that testicular IL-l specifically originates from Sertoli cells. The assay used for IL-l is known to detect IL-la and IL-l/3 as well as IL-2 (Dinarello, 1985; ~pp~nheim et al., 1986). No IL-2 could be detected in SCCM. The fact that the activity measured here was specificaIly blocked by IL-la antiserum demonstrates that Sertoli cell produces IL-1 cy. Furthermore our results also demonstrate that Sertoli cell IL-la secretion increases with sexual maturation; IL-la was first detected in Sertoli cell cultures prepared from 35-day-old donors and markedly rose between 35 and 45 days. This is in keeping with the previous findings of Syed et al. (1988) showing that IL-I production by cultured seminiferous tubule also develops in parallel to sexual maturation. The biological role of testicular IL-l ac is still obscure. However, the results presented here are relevant to the recent observations showing that IL-I receptors may be localized on germ cells (Takao et al., 1990) and that IL-la stimulates spermatogonial mitosis of hypophysectomized rats in vivo (PGlIBnen et al., 1989) as well as spermatogenial and meiotic DNA synthesis in cultured isolated seminiferous tubules (Parvinen et al., I991; Slider et al., 1991). Therefore, these observations and the result of this study support the hypothesis that IL- I a may play an important role in the paracrine regulation of germ cells by Sertoli cells.
Dinarelto, CA. (19%) Immunobiology t72. 301-315. Galdieri, M., Ziparo, E., Palombi, F., Russo, M.A. Stephanini, M. (1981) J. Andrul. 5, 249-259. Gillis,
S., Ferm,
Immunol. Jigou.
R.,
M.M..
Ou.
W.
and Smith.
Le
Magueresse,
J.
P.. Pinenu, C., D.H., Guillou. F. and
B., Sourdaine,
Boisseau, C. (1988)
in Molecular
and Cellular
ogy of the Testis, Vol. 50 (Cooke, eds.), pp. 225-270, S.A.,
Slider,
Endocrinol-
B.A. and Sharpe.
R.M.,
Raven Press. New York. 0..
and Ritz&n. E.M.
Syed. V.,
(1987)
Khan. S.A.. Schmidt.
Gustafsson,
lnt. J. And&.
K., Lindh,
E. (19881
Mol.
M.
10, 495 -503.
Geyter,
K.. Wallin. P,, Di Pauli, R.. De
Ch. and Nieschlag, 22
(197X)
120. 2027-2032.
Velez de la Calle. J.F., Gamier,
Khan.
K.A.
and
Cell.
Endocrinol.
58,
l-230.
Le Magu~ress~, ih72-
B. and J&gou, B. (1988)
~nd~~cr~n~~l~gy 122,
IhHO.
Mather,
J.P. and Phillips. D.M.
free Culture D.W..
flY84)
in Methods
of Cells of the Endocrine
Sirbasku.
D.A.
and Sato,
for Serum-
System tBarnes,
G.H..
eds.),
pp. 29-45,
W.A..
Grimes,
Alan R. Liss. New York. Meistrich,
M.L..
Mace, M.L. Oppenheim,
the
Immunol.
M.. SGder-. 0. 11th North
Pursis.
Dev.
and Ritz&,
0.
K. and Durum.
E.M.
Testis
tL991)
Program
Workshop.
of
Montreal,
and
Parvinen.
M.
(19891
Reprod.
V. (1978)
Int.
I, 85-87.
K., Clausen,
Androl.
Matsushima,
91.
P.. Slider,
Fertil.
S.R. xnd
25, 1065-1077.
Today 7. 4.5-X
American
1991. p. 34 (Ahstr. Piiiilnen,
J., Brock.
Biol. Reprod.
J.J.. Kovacs. E.J..
S.K. (19%) Parvinen,
Longtin, (1981)
O.P.F.
and Hansson.
J.
2. 342-352.
Scala. C. and Oppenheim,
J.J. (1983) J. lmmunol.
131, IlhO-
11hti. R.M. (19%)
Sharpe. Slider,
0..
Pawinen, Androi. Syed, V., Ritzin, Takao,
Clin. ~ndo~rjn[)i.
Syed. V.. Callard, M.,
Friiysa.
Metah.
Toppari,
B. and Ritz&,
15. 185-207. J., Piill%nen+ P.,
E.M.
(1991)
%der, 0.. Arver, S.. Lindh. M.. Khan, E.M. (19881 hit. J. Androl. II, 437-437.
T., Mitchell,
Toebosch,
fnt. J.
14. Z-231.
W.M.,
(1990) Endocrinology F.H.
G.V..
A.M.W., and
Tracey,
and De Souza, E.B.
127, 25 i -258.
Robertson,
Grootegoed.
D.E.
S. and
D.M.,
J.A.
(1989)
K&i,
LA.,
De Jong,
J. Endocrinol.
121,.
X7-762.
We are grateful to Dr. S. Poole (NBSB, Hertfordshire, U.K.) for the sheep anti-murine IL-lcu
Valotaire.
Y. and Duval,
1211-1224.
J. (1969)
Bull. Sot. Chim.
Biol. 51,
MOLCEL
R13 l/$03.50
02665
Rapid Paper
Sertoli cells are the site of interleukin-lcu Nadine, G&rard
synthesis in rat testis
‘, Viqar Syed I-2, Wayne Bardin 2, NoElle Genetet and Bernard
’
Jkgou ’
’ G. E.R.M., INSERM CJF 91-04, Unilvmitr: de Rennrs I, 35042 Rmne.s, France, ’ The Population Council, New York, NY 10021, U.S.A., und .’ G. U. R. I. F.A., 35036 Rennes, France (Received
Key words: Interleukin-1;
Sertoli
11 September
cell: Spermatogenesis;
199 1; accepted
Paracrine
factor;
16 September
199 1)
(Rat testis)
Summary To elucidate the cellular origin of interleukin-1 (IL-11 in the mammalian testis, we assayed IL-1 activity in culture media of Sertoli cells collected from rats at 20, 35 and 45 days of age as well as in culture media of interstitial, peritubular and germ cells from adult rats. IL-l was detected in Sertoli cell culture media isolated from 35 and 45-day-old rats. At 45 days approximatly twice as much IL-l was produced than at 35 days. In contrast, IL-1 activity was not detected in 20-day-old rat Sertoli cell culture media nor in interstitial, peritubular and germ cell culture media. The Sertoli cell IL-1 activity was specifically neutralized by an IL-la antiserum. It is concluded that Sertoli cells produce IL-la and that this production increases during sexual maturation.
Introduction The mammalian seminiferous tubules are comprised of the somatic Sertoli cells and germ cells whose proliferation is under the hormonal control of LH and FSH as well as of an extremely complex set of paracrine factors (Sharpe, 1986; Jtgou et al., 1988). One of them, an interleukinla-like factor, was identified in the testis of the rat after 30 days of age (Khan et al., 1987; Syed et al., 1988) and in testicular extracts of the adult human (Khan et al., 1988). Using indirect approaches, Khan et al. (1987) and Syed et al. (1988) have suggested that Sertoli cells may be
Address for correspondence: SERM CJF 91-04, UniversitC Beaulieu, 35042 Rennes Cedex,
B. JCgou. de Rennes France.
G.E.R.M., I, Campus
INde
the source of the testicular interleukin-la (IL-la) production, but to date there is no direct evidence to substantiate this hypothesis. In the present study we demonstrate that rat Sertoli cells in vitro specifically secrete IL-la and that the production of IL-la increases with the stages of sexual development. Materials
and methods
Sertoli cell isolation and culture. Sertoli cells were prepared from 20-, 35- and 45-day-old Sprague-Dawley rats according to methods described elsewhere (Mather and Phillips, 1984; Toebosch et al., 1989) and cultured at 32°C in a humidified atmosphere of 5% CO,-95% air in Ham’s F12/DMEM (v/v; Gibco BRL) supplemented with insulin (10 pg/mll, transferrin (5 pg/ml) and gentamicin (4 pg/ml). After 2-3
RI4
Germ cell isolation and culture. Germ cells prepared from adult Sprague-Dawley rat testes by trypsinisation (Meistrich et al., 1981) were submitted to a centrifugal elutriation. The two fractions obtained: pachytene spermatocytes and round spermatids (90% enrichment) were incubated in Ham’s FIZ/DMEM (v/v) enriched with lactate (6 mM), pyruvate (2 mM) and gentamicin (4 ~g/ml). After 24 h of culture the media were kept at -80°C. Interstitial and peritubular cell isolation and culture. Interstitial and peritubular cells were isolated from 20- and from W-day-old SpragueDawley rat testes by collagenase digestion according to methods previously described (Purvis et al.,
days of culture, Sertoli cell monolayers were exposed to a Tris-HCI buffer solution (20 mM, pH 7.4) to remove contaminating germ cells (Galdieri ct al., 1981; Le Magueresse and Jegou, 1988). The day after, cells were incubated for an additional 24 h in medium where transferrin was omitted. The media were kept at -8c1”C and the cells were scraped and frozen at -80 “C until DNA content assay (Valotaire and Duval, 1969). At the termination of the culture, the purity of the Sertoli cell cultures was determined: perituhular cell and germ cell contaminations were less than 7% and 2% respectively for 20-day-old Sertoli cells, 3% and 7% respectively for Sertoli cells from older animals.
/.:
20
35
45
AGE
-I-rl
C PHA
32 16
8
4
2
16
8
4
2
16
20 Days L-IL-la!
curves of IL-I
C: control cells without PHA.
2
35 oays
DILUTIONS
activity obtained with human recombinant Inset: IL-la
4
-J RECIPROCAL
Fig. 1. Dose-response
8
IL-la
and SCCM
from 20-. 35 and 45day-old-rats.
secretion expressed in units per gg of Sertoli cell DNA
at the different
ages.
R15
1978; Mather and Phillips, 1984). The cells were washed to remove most of the contaminating cells after 1 h of preincubation. They were cultured for 24 h and the media kept at - 80 o C. Bioassay of IL-I. IL-l was measured in the different culture media using the murine thymocyte proliferation assay (Scala and Oppenheim, 19831, with minor modifications. Briefly, thymocytes (15 x 10’ ceils/ml) from 4-&week-old CH,/HE female mice were incubated in the presence of phytohemaglutinin (PHA, 20 pg/ml), with various doses of human recombinant IL-la (Boehringer-Mannheim; starting dilution: 50 U/ml> or test material, for 48 h at 37°C. During the last 2 h of incubation [‘Hlthymidine was added. Incorporated radioactivity was determined by liquid scintillation counting and results were expressed as mean counts per min (cpm> of triplicate cultures. In our assay conditions, one unit of IL-1 activity was arbitrarily defined as the ability of test material to double [~H]thymidine incorporation into thymocytes, when compared with PHA stimulated cultures (Syed et al., 1988).
El
scM
m
scLY4 + ANTI IL-la
m
SCCM + ANTI IL-l@
Bioassay of IL-2. IL-2 activity was assayed using a specific bioassay based on the measurement of the proliferation of a cloned murine cytotoxic T lymphocyte line (CTLL 2) (Gillis et al., 1978). Results As shown in Fig. 1, Sertoli cell culture media (SCCM) obtained from 35- and 45-day-old-rats induced a significant dose-dependent proliferation of murine thymocytes, whereas no IL-l activity was detected in the culture media of Sertoli cells prepared from ZO-day-old animals. Germ cell conditioned media as well as interstitial and peritubular cell culture media lacked IL-l activity (data not shown). It is noteworthy that SCCM prepared with cells collected from pubertal rats f45-day-old) displayed approximatively twice as much bioactivity as SCCM prepared with cells collected from prepubertal animals (35-day-old). Of note also is the fact that while the specific IL-la antiserum could block the bioactivity of the
7
.L. . . . . . . . . . .
. . . . .
. . . . .
-! I)... . .
. . ..*
.
.
.
.
.
. .
. .
. .
. .
.
.
.
.
.
.
.
.
a... . . . . . . . . .*..
. . . .
. . . .
..,.
_
45 Days PHA
Fig. 2. Effect of sheep anti-murine
IL-la
1
SCCti
1
and IL-l@ antisera on the bioactivity of IL-I. SCCM were incubated h at room temperature before being assayed.
with the antibodies
1
SCCM prepared from the older Sertoli cell donors, the specific IL-ID antiserum had no effect (Fig. 2). Moreover no IL-2 bioactivity could be detected in any of the media tested (data not shown).
and IL-l@ antisera. This work was supported by I’INSERM (Paris, No. 900406), la Fondation de France (Paris) and NIH grant HD 13541.
References Discussion
Our data confirm previous results showing that peritubular and interstitial cells do not secrete IL-1 Khan et al., 1987; Syed et al., 1988). Therefore, the data reported here demonstrate that testicular IL-l specifically originates from Sertoli cells. The assay used for IL-l is known to detect IL-la and IL-l/3 as well as IL-2 (Dinarello, 1985; ~pp~nheim et al., 1986). No IL-2 could be detected in SCCM. The fact that the activity measured here was specificaIly blocked by IL-la antiserum demonstrates that Sertoli cell produces IL-1 cy. Furthermore our results also demonstrate that Sertoli cell IL-la secretion increases with sexual maturation; IL-la was first detected in Sertoli cell cultures prepared from 35-day-old donors and markedly rose between 35 and 45 days. This is in keeping with the previous findings of Syed et al. (1988) showing that IL-I production by cultured seminiferous tubule also develops in parallel to sexual maturation. The biological role of testicular IL-l ac is still obscure. However, the results presented here are relevant to the recent observations showing that IL-I receptors may be localized on germ cells (Takao et al., 1990) and that IL-la stimulates spermatogonial mitosis of hypophysectomized rats in vivo (PGlIBnen et al., 1989) as well as spermatogenial and meiotic DNA synthesis in cultured isolated seminiferous tubules (Parvinen et al., I991; Slider et al., 1991). Therefore, these observations and the result of this study support the hypothesis that IL- I a may play an important role in the paracrine regulation of germ cells by Sertoli cells.
Dinarelto, CA. (19%) Immunobiology t72. 301-315. Galdieri, M., Ziparo, E., Palombi, F., Russo, M.A. Stephanini, M. (1981) J. Andrul. 5, 249-259. Gillis,
S., Ferm,
Immunol. Jigou.
R.,
M.M..
Ou.
W.
and Smith.
Le
Magueresse,
J.
P.. Pinenu, C., D.H., Guillou. F. and
B., Sourdaine,
Boisseau, C. (1988)
in Molecular
and Cellular
ogy of the Testis, Vol. 50 (Cooke, eds.), pp. 225-270, S.A.,
Slider,
Endocrinol-
B.A. and Sharpe.
R.M.,
Raven Press. New York. 0..
and Ritz&n. E.M.
Syed. V.,
(1987)
Khan. S.A.. Schmidt.
Gustafsson,
lnt. J. And&.
K., Lindh,
E. (19881
Mol.
M.
10, 495 -503.
Geyter,
K.. Wallin. P,, Di Pauli, R.. De
Ch. and Nieschlag, 22
(197X)
120. 2027-2032.
Velez de la Calle. J.F., Gamier,
Khan.
K.A.
and
Cell.
Endocrinol.
58,
l-230.
Le Magu~ress~, ih72-
B. and J&gou, B. (1988)
~nd~~cr~n~~l~gy 122,
IhHO.
Mather,
J.P. and Phillips. D.M.
free Culture D.W..
flY84)
in Methods
of Cells of the Endocrine
Sirbasku.
D.A.
and Sato,
for Serum-
System tBarnes,
G.H..
eds.),
pp. 29-45,
W.A..
Grimes,
Alan R. Liss. New York. Meistrich,
M.L..
Mace, M.L. Oppenheim,
the
Immunol.
M.. SGder-. 0. 11th North
Pursis.
Dev.
and Ritz&,
0.
K. and Durum.
E.M.
Testis
tL991)
Program
Workshop.
of
Montreal,
and
Parvinen.
M.
(19891
Reprod.
V. (1978)
Int.
I, 85-87.
K., Clausen,
Androl.
Matsushima,
91.
P.. Slider,
Fertil.
S.R. xnd
25, 1065-1077.
Today 7. 4.5-X
American
1991. p. 34 (Ahstr. Piiiilnen,
J., Brock.
Biol. Reprod.
J.J.. Kovacs. E.J..
S.K. (19%) Parvinen,
Longtin, (1981)
O.P.F.
and Hansson.
J.
2. 342-352.
Scala. C. and Oppenheim,
J.J. (1983) J. lmmunol.
131, IlhO-
11hti. R.M. (19%)
Sharpe. Slider,
0..
Pawinen, Androi. Syed, V., Ritzin, Takao,
Clin. ~ndo~rjn[)i.
Syed. V.. Callard, M.,
Friiysa.
Metah.
Toppari,
B. and Ritz&,
15. 185-207. J., Piill%nen+ P.,
E.M.
(1991)
%der, 0.. Arver, S.. Lindh. M.. Khan, E.M. (19881 hit. J. Androl. II, 437-437.
T., Mitchell,
Toebosch,
fnt. J.
14. Z-231.
W.M.,
(1990) Endocrinology F.H.
G.V..
A.M.W., and
Tracey,
and De Souza, E.B.
127, 25 i -258.
Robertson,
Grootegoed.
D.E.
S. and
D.M.,
J.A.
(1989)
K&i,
LA.,
De Jong,
J. Endocrinol.
121,.
X7-762.
We are grateful to Dr. S. Poole (NBSB, Hertfordshire, U.K.) for the sheep anti-murine IL-lcu
Valotaire.
Y. and Duval,
1211-1224.
J. (1969)
Bull. Sot. Chim.
Biol. 51,