CONTRACEPTION
PROGESTERONE
LEVELS
Pr-D-hCG-TT AND hCG
S.
IN
AFTER DURING
MONKEYS
C.
Ramakrishnan,
Department
of
Institute
of
Il”iMUNIZED
INJECTION OF LUTEAL PHASE
Das
&
G.P.
Biochemistry, [Yledical
WITH
hLH
Talwar
A11
Sciences,
India New
Delhi,
India
ABSTRACT
Rhesus monkeys were injected
Pr-$-hCG-TT phase
of
the
biological neutralized monkeys
cycle
in
immunized with order
activity of by circulating did
not
show
levels following one circulating antibodies increase
in
plasma
a rise or
to
these
with hCG/hLH check
the in
vaccine the luteal
whether
the
gonadotropins antibodies. in
plasma
was The immunized progesterone
more injections did not, however,
progesterone
levels
administered.
Accepted
JULY
for
publication
1978 VOL. 18 NO. I
Zune
12,
1978
of
hCG.
prevent when hLH
The an was
CONTRACEPTION
INTRODUCTION
In a preceding paper (1) we have reported the ability of the antisera generated by the vaccine Pr-B-hCG-TT to neutralize the hCG induced steroidogenesis in mouse Leydig cells and gonadotropin induced ovulation in mice. This paper deals with studies on the discriminatory capacity of the circulating antibodies to interfere in the biological action of the pituitary and placental gonadotropins on the primate ovaries -in viva. Rhesus monkeys immunized with the vaccine Pr-$-hCG-TT were injected with hLH and/or hCG in the late luteal phase and the biological activity of the hormones was assessed by measuring the plasma progesterone levels.
WTERIALS
AND
METHODS
Animals : Five regularly cycling monkeys were immunized with the vaccine Pr-B-hCG-TT as described earlier (2). rs were followed by The anti-hCG ti1S51_hCG bound in estimating the percentage of The antiappropriately diluted sera samples (3). tetanus toxoid titers were measured by passive haemaqglutination assay (2). Menstrual cycle of monkeys were followed by daily vaginal smears and periodic assessment of plasrna progesterone levels in the luteal phase.
Hormone
administration
:
Highly purified human lutenizing hormone (hLH;5000 IU/mg) was kindly made available by Dr. B.B. Saxena, Cornell University [Yledical College, New York. hLH was dissolved in phosphate buffered saline (pH 7.4) and administered through the saphenous vein or hCG (10,000 IU/mg) provided by intramuscularly. Dr. Tsong of Population Council, New York, was Blood samples were drawn injected intramuscularly. in heparinized tubes from sites contralateral to that of injection. Plasma separated after centrifugation, was stored at -20°C.
52
JULY
1978 VOL. 18 NO. I
CONTRACEPTION
Proqesterone
estimation
:
Plasma progesterone levels were estimated by radioimmunoassay (4). The sensitivity range of the assay was between 20 pg to 320 pg.
RESULT5
AND
COMMENTS
plankey 5 was immunized with Pr-p-hCG-TT 25 imonths prior to this stud It had an antibody titer of 32 76 binding of 1% I-hCG at 1:3200 dilution. The average cycle length was 23 days. The monkey was given an intravenous injection of 75 IU of hLH on day 17 of the cycle. Blood samples removed at different time periods following hLH administration showed an elevation of plasma progesterone. Peak values were noted in blood sample taken at 24 hours (Fig. '!). Similar results were obtained in another monkey (No. 8) in which hLH (180 IU) was injected in three doses on successive days intramuscularly. On the fifth day after hLH injection, monkey 5 was given 600 IU of hCG and again twice this amount (1200 IU) on the following day. C!onkey 8 received d single injection of 1200 IO of hCG on day 6 after hLH challenge. No rise in progesterone values were noted following these injections. The idck of rise in progesterone following hCG injections could be due to the blocking effect of the circulating antihCG antibodies. Yowever, this may also be due to the corpu;
i.uteurn
cxpcsu:
e
to
becozlnl,
In another ,dlth
?
refractory
jon~dotroo2ns
circuldtln9
monkey
aii
reF.eat-ed
(5). iwnunLred
5
:ICT th;
eai-lL-:r
dntlbody titer of 44' 3~~ 1:3?C dilution, hCG .Q.JSqlven instedd of >hLh. l'iO0 IU of nCG 'was injected an 3 doses ;it 24-hqur intzrvdl. The plasma prn(;esterone l-wels did not shur: any incredse .Ln spite 3f ddec/tidte l:lst1lldtic? Jf hCG Simllclr results here obtd-ned ':_ monkeys (Fig. 2). '~ters ::f 45.7@,i 0 and ;' with clrcul,~t~ng dnt:-hCG .
JULY
197X VOL.
1X .NO. 1
CONTRACEPTION
54
JULY
1978 VOL. 18 NO. 1
CONTRACEPTION
UI CG OODJ
KG 5oon.J
1
JULY
1978
VOL. 18 NO. I
hCG 5oonJ
1
CONTRACEPTION
, l
-40
- 20
0
10
TIME
56
20
IN
1
.
30
40
,
50
#C.”
60
96
HOURS
JULY
1978 VOL. 18 NO. 1
CONTRACEPTION
at 1:3200 and 24.6$ fairly high amounts plasma progesterone
at 1:320, where hCG did not provoke the levels (Fig. 2).
given in rise in
The hCG used in these studies was biologically active as tested in the Leydig cell assay, induction of ovulation in mouse and mouse uterine weight gain I-\control non-inimunized monkey (No. 36) dssays. injected with 200 IIJ of IhCG intramuscularly showed a rise in the plasma progesterone values (Fig. 3). have shown that hLH/hCG Besides, Stouffer -et al.(6) increases the progesterone production in cultured Luteal cells from rhesus monkey.
These studies demonstrate the effectiveness of the antibodies in rhesus monkeys imriiunized with the vaccine Pr-p-hCG-TT to counteract the action of hCG on ovarian steroldogenesis in the luteal phase of the cycle -in viva. The antibodies, however, failed to prevent the hLH induced rise in the orogesterone secretion.
This work was supported by grants from the International Committee fcr Contraception Fiesedrch of the Population Council, New York, The Reeearch h Training Center grant of 'World Health Organization, the 1.D.R.C. of Canada 8 The Family Planning Foundation of India. We thank Shri Gurbdchan Sinqh and Sri Chand for their tochnicdl dssi:;cance.
JULY
1978VOL.18N0.1
57
CONTRACEPTION
REFERENCES 1.
Das, C., Talwar, G.P., Rdmakrishnan, S., Salahuddin, PI., Kumar, S., Hingoranr, V. and Coutinho, E., Croxatto, He, Hemmingson, E., Johansson, E., Luukkainen, T. dnd Shahani, 5.: Discriminatory effect of anti-Pr-B-hCG-TT antibodies on the neutralization of biological activity of olacental and pituitary gonadotropins (communicated ; n the presP?t. series) +
2.
Talwar, G.P., Dubey, S.K., Salahuddin, 14. and Shastri, N..: Kinetrcs of antibody response in animals injected with processed beta-hCG conjugated to tetanus toxoid (Pr-B-hCG-TT). Contraception 13: 153 11976'.
3.
Dubey, S.K., Talwar, 5.P. dinitrophenyl of the hCG.
4.
Guerrero, R., Cekan, i. and Hso, T., A rapid 5-hour radioimmunoDiczfalusy, E.: assay of prooesterone and oestradiol in human Eli;. 4: 173 (1975'. Endocrinol. plasma.
5.
Neil, of
6.
the
J.D.
Salahuddin, M., Shastri, ti. and : immunological properties of derivatives of beta-subunit Contraception 13: 141 :1976!.
and
Knobil,
initial
nancy
in
the
90:34
!1972'.
On
E.:
luteotropic rhesus
the
stimulus
nature of
preg-
Endocrinology
monke,y.
Stouffer, R.L., Nixon, M.E., Gulyas, B-J. Gonadotropin sensitive and Hodgen, G.D.: progesterone production by rhesus monkey iuteal of
cells the
cycle*
corpus
in --
vitro luteum
Endocrinology
: during 100:
A
function the 506
of
age
menstrual (lY77‘.
JULY
1978 VOL.18
NO.1
PROGESTERONE
LEVELS
Pr-D-hCG-TT AND hCG
S.
IN
AFTER DURING
MONKEYS
C.
Ramakrishnan,
Department
of
Institute
of
Il”iMUNIZED
INJECTION OF LUTEAL PHASE
Das
&
G.P.
Biochemistry, [Yledical
WITH
hLH
Talwar
A11
Sciences,
India New
Delhi,
India
ABSTRACT
Rhesus monkeys were injected
Pr-$-hCG-TT phase
of
the
biological neutralized monkeys
cycle
in
immunized with order
activity of by circulating did
not
show
levels following one circulating antibodies increase
in
plasma
a rise or
to
these
with hCG/hLH check
the in
vaccine the luteal
whether
the
gonadotropins antibodies. in
plasma
was The immunized progesterone
more injections did not, however,
progesterone
levels
administered.
Accepted
JULY
for
publication
1978 VOL. 18 NO. I
Zune
12,
1978
of
hCG.
prevent when hLH
The an was
CONTRACEPTION
INTRODUCTION
In a preceding paper (1) we have reported the ability of the antisera generated by the vaccine Pr-B-hCG-TT to neutralize the hCG induced steroidogenesis in mouse Leydig cells and gonadotropin induced ovulation in mice. This paper deals with studies on the discriminatory capacity of the circulating antibodies to interfere in the biological action of the pituitary and placental gonadotropins on the primate ovaries -in viva. Rhesus monkeys immunized with the vaccine Pr-$-hCG-TT were injected with hLH and/or hCG in the late luteal phase and the biological activity of the hormones was assessed by measuring the plasma progesterone levels.
WTERIALS
AND
METHODS
Animals : Five regularly cycling monkeys were immunized with the vaccine Pr-B-hCG-TT as described earlier (2). rs were followed by The anti-hCG ti1S51_hCG bound in estimating the percentage of The antiappropriately diluted sera samples (3). tetanus toxoid titers were measured by passive haemaqglutination assay (2). Menstrual cycle of monkeys were followed by daily vaginal smears and periodic assessment of plasrna progesterone levels in the luteal phase.
Hormone
administration
:
Highly purified human lutenizing hormone (hLH;5000 IU/mg) was kindly made available by Dr. B.B. Saxena, Cornell University [Yledical College, New York. hLH was dissolved in phosphate buffered saline (pH 7.4) and administered through the saphenous vein or hCG (10,000 IU/mg) provided by intramuscularly. Dr. Tsong of Population Council, New York, was Blood samples were drawn injected intramuscularly. in heparinized tubes from sites contralateral to that of injection. Plasma separated after centrifugation, was stored at -20°C.
52
JULY
1978 VOL. 18 NO. I
CONTRACEPTION
Proqesterone
estimation
:
Plasma progesterone levels were estimated by radioimmunoassay (4). The sensitivity range of the assay was between 20 pg to 320 pg.
RESULT5
AND
COMMENTS
plankey 5 was immunized with Pr-p-hCG-TT 25 imonths prior to this stud It had an antibody titer of 32 76 binding of 1% I-hCG at 1:3200 dilution. The average cycle length was 23 days. The monkey was given an intravenous injection of 75 IU of hLH on day 17 of the cycle. Blood samples removed at different time periods following hLH administration showed an elevation of plasma progesterone. Peak values were noted in blood sample taken at 24 hours (Fig. '!). Similar results were obtained in another monkey (No. 8) in which hLH (180 IU) was injected in three doses on successive days intramuscularly. On the fifth day after hLH injection, monkey 5 was given 600 IU of hCG and again twice this amount (1200 IU) on the following day. C!onkey 8 received d single injection of 1200 IO of hCG on day 6 after hLH challenge. No rise in progesterone values were noted following these injections. The idck of rise in progesterone following hCG injections could be due to the blocking effect of the circulating antihCG antibodies. Yowever, this may also be due to the corpu;
i.uteurn
cxpcsu:
e
to
becozlnl,
In another ,dlth
?
refractory
jon~dotroo2ns
circuldtln9
monkey
aii
reF.eat-ed
(5). iwnunLred
5
:ICT th;
eai-lL-:r
dntlbody titer of 44' 3~~ 1:3?C dilution, hCG .Q.JSqlven instedd of >hLh. l'iO0 IU of nCG 'was injected an 3 doses ;it 24-hqur intzrvdl. The plasma prn(;esterone l-wels did not shur: any incredse .Ln spite 3f ddec/tidte l:lst1lldtic? Jf hCG Simllclr results here obtd-ned ':_ monkeys (Fig. 2). '~ters ::f 45.7@,i 0 and ;' with clrcul,~t~ng dnt:-hCG .
JULY
197X VOL.
1X .NO. 1
CONTRACEPTION
54
JULY
1978 VOL. 18 NO. 1
CONTRACEPTION
UI CG OODJ
KG 5oon.J
1
JULY
1978
VOL. 18 NO. I
hCG 5oonJ
1
CONTRACEPTION
, l
-40
- 20
0
10
TIME
56
20
IN
1
.
30
40
,
50
#C.”
60
96
HOURS
JULY
1978 VOL. 18 NO. 1
CONTRACEPTION
at 1:3200 and 24.6$ fairly high amounts plasma progesterone
at 1:320, where hCG did not provoke the levels (Fig. 2).
given in rise in
The hCG used in these studies was biologically active as tested in the Leydig cell assay, induction of ovulation in mouse and mouse uterine weight gain I-\control non-inimunized monkey (No. 36) dssays. injected with 200 IIJ of IhCG intramuscularly showed a rise in the plasma progesterone values (Fig. 3). have shown that hLH/hCG Besides, Stouffer -et al.(6) increases the progesterone production in cultured Luteal cells from rhesus monkey.
These studies demonstrate the effectiveness of the antibodies in rhesus monkeys imriiunized with the vaccine Pr-p-hCG-TT to counteract the action of hCG on ovarian steroldogenesis in the luteal phase of the cycle -in viva. The antibodies, however, failed to prevent the hLH induced rise in the orogesterone secretion.
This work was supported by grants from the International Committee fcr Contraception Fiesedrch of the Population Council, New York, The Reeearch h Training Center grant of 'World Health Organization, the 1.D.R.C. of Canada 8 The Family Planning Foundation of India. We thank Shri Gurbdchan Sinqh and Sri Chand for their tochnicdl dssi:;cance.
JULY
1978VOL.18N0.1
57
CONTRACEPTION
REFERENCES 1.
Das, C., Talwar, G.P., Rdmakrishnan, S., Salahuddin, PI., Kumar, S., Hingoranr, V. and Coutinho, E., Croxatto, He, Hemmingson, E., Johansson, E., Luukkainen, T. dnd Shahani, 5.: Discriminatory effect of anti-Pr-B-hCG-TT antibodies on the neutralization of biological activity of olacental and pituitary gonadotropins (communicated ; n the presP?t. series) +
2.
Talwar, G.P., Dubey, S.K., Salahuddin, 14. and Shastri, N..: Kinetrcs of antibody response in animals injected with processed beta-hCG conjugated to tetanus toxoid (Pr-B-hCG-TT). Contraception 13: 153 11976'.
3.
Dubey, S.K., Talwar, 5.P. dinitrophenyl of the hCG.
4.
Guerrero, R., Cekan, i. and Hso, T., A rapid 5-hour radioimmunoDiczfalusy, E.: assay of prooesterone and oestradiol in human Eli;. 4: 173 (1975'. Endocrinol. plasma.
5.
Neil, of
6.
the
J.D.
Salahuddin, M., Shastri, ti. and : immunological properties of derivatives of beta-subunit Contraception 13: 141 :1976!.
and
Knobil,
initial
nancy
in
the
90:34
!1972'.
On
E.:
luteotropic rhesus
the
stimulus
nature of
preg-
Endocrinology
monke,y.
Stouffer, R.L., Nixon, M.E., Gulyas, B-J. Gonadotropin sensitive and Hodgen, G.D.: progesterone production by rhesus monkey iuteal of
cells the
cycle*
corpus
in --
vitro luteum
Endocrinology
: during 100:
A
function the 506
of
age
menstrual (lY77‘.
JULY
1978 VOL.18
NO.1
