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Inhibition of ovulation in the rat by a hCG antagonist
CONTRACEPTION
INHIBITION
OF OVULATION
K.
IN
Kato
THE
and
RAT
M.R.
BY A hCG ANTAGONIST
Sairam
Reproduction Research Laboratory Clinical Research Institute of Montreal 110
Pine
Montreal,
Avenue
West
Quebec Canada
H2W lR7
ABSTRACT
Chemically shown
deglycosylated
to
have
pete with pregnant
1251-labeled rats. DG-hCG
PMSG-treated with
25
hormonal
hCG
its
antagonistic
hormonal
Submitted
MAY
hCG for binding at 100 ug dose
Administration reduced ovulation
was capable proestrus.
for for
publication publication
1983VOL.27NO.S
of
of
March
in
DG-hCG prior (P < 0.05).
in
ovulation indicate viwv
7,
1983
29,
1983
in
which were
the ovary ovulation
37%
by
activity
(DG-hCG) LM wtia
sites inhibited
of inhibiting These results
April
hCG
activity
rats.
IU
rats, DG-hCG afternoon of
Accepted
preparations antagonistic
to
have able
in by
been to
com-
pseudo73% in
and concurrent In mature cycling
when injected on the that DG-hCG can exert
rats.
515
CONTRACEPTION
INTRODUCTION Human choriogonadotropin (hCG) is a dimeric glycoprotein hormone whose However, the role chemical and biological properties are well known. of the bulky carbohydrate groups which comprise up to 35% of the molecule In a recent study, we showed that chemical has not been fully clarified. deglycosylation of hCG by anhydrous hydrogen fluoride treatment (HF) provided a hormone derivative with about 75% less carbohydrate (1). The modification which did not affect the polypeptide moiety of the hormone including its subunit structure had discordant effects on the biological properties of the hormone (l,Z), a feature with interesting potentials for elucidating its mechanism of action. The deglycosylated hCG (DG-hCG) which had an enhanced affinity for the hormone receptors in the testis and ovary and retained its immunological activity was virtually incapable of inducing hormone response in gonadal cells incubated ill utiu (l,Z). It was further demonstrated that the DG-hCG was an effective antagonist of the action of hCG or LH in W&W by inhibiting hormone-induced cyclic AMP accuSince DG-hCG virtually lacks sialic mulation and steroidogenesis (2). acid which is thought to be responsible for the relatively long half-life of hCG in circulation in rats (3), it was of interest to evaluate the antawe have investigated the gonistic activity in wivo. In the present study, effect of DG-hCG on ovulation using immature and mature rats.
MATERIALS
AND METHODS
hCG was purified from the commercially available crude powder and shown to have chemical and biological properties similar to a NIAMDDK preparation This was used for the preparation of deglycosylated hCG (DG-hCG) (CR 119). The dry hormone powder treated with hydrogen as described recently (1). fluoride for 60 mins at O°C was purified on concanavalin-A sepharose and sephadex G-100 (1). The DG-hCG was obtained as a stable lyophilized product which was stored at 4OC. Asialo hCG was prepared by treatment with immobilized neuraminidase (Sigma) and lyophilized.
Immature (40-45 Dawley CD rats were maintained 0500 h to 1900
g at 20 days of age) and adult (200-250 g) female Spraguewere purchased from Charles River Canada, Montreal. All rats on a 14-hr light - IO-hr dark lighting schedule (lights h) with food and wate.r ad LibLtum.
Inhibitian 06 upLake
06 lz51-hCG by VG-hCG
Immature female rats were made pseudopregnant by administration of 25 IU of PMSG on the 25th day followed by an injection of 25 IU of hCG 56-65 hrs later. The animals were used between days 6-8 after this treatment. The ability of unlabeled hCG, DG-hCG or asialo hCG to compete for binding sites for ‘251-hCG in the ovary was tested by administering them simultaneously. Two hrs after their intracardiac administration, the amount of radioactivity in the ovary and blood was determined and expressed as a ratio.
516
MAY
1983 VOL. 27 NO. 5
CONTRACEPTION Induction
o6 ovulation
Immature rats were maintained for 5 days and treated subcutaneously with The experimental schedule was identi25 Iu of PMsG (NIAMDDK, 1790 IU/mg). cal to that reported earl ier from our laboratory (4). Two days after PMSG, the DG-hCG or vehicle was administered subcutaneously with 50% of the dose distributed at 45 hrs and 48 hrs, respectively. This was followed by subcutaneous injection of 25 IU of hCG or vehicle at 48 hrs after PMSG. The animals were sacrificed under ether anesthesia 17-18 hrs after the last hormone injection and the oviducts were dissected. The number of ova released into the oviduct were examined and counted under the microscope as described previously (4). In this experiment PMSG and hCG solutions were prepared in saline. DG-hCG solution was prepared in 2% gelatin (Eastman Chemicals) with 0.9% NaCi and adjusted to pH 7.5 using IN NaOH. The effect of DG-hCG on ovulation in cycling rats was also tested by administering it on the afternoon of proestrus and examining for ova in the oviduct on the following day (see Table Ill). The
results
were
analyzed
for
statistical
significance
by Student’s
‘t’
test
RESULTS hCG and DG-hCG were effective in inhib As shown in Table I, both unlabeled ing the accumulation of 1251-labeled hCG in the pseudopregnant rat ovary. In marked contrast to this, up to 25 pg of asialo hCG was totally ineffect in competing with labeled hCG for binding sites in the ovary. TABLE
Ghoup
I:
Th&uinent '251-hCG p.Lw UvLeabeRed
I
Vehicle
2
hCG, 5 ug DG-hCG, 5.7 ug DG-hCG, 11.4 ug Asialo hCG, 25 ug
;
ive
INHIBITION OF UPTAKE OF lz51-hCG IN THE PSEUDOPREGNANT RAT BY UNLABELED HORMONES
----_-_-_-_-_
5
it-
Ratio
06 hU&Oativ~y
-in 100 mg ovw~q/lOO UP bLood Mean
120.0 .23.0 ill.0 19.0 101.6
* SEM
+ _+ * f *
6.0 0.6 2.3 3.6 9.8
P < 0.05 NS P < 0.05 NS
ng egtivafknt 06 lz51-hCG (npec&jicactivMy 60-90 i_~Ci/ into p~a~dopheynani htis (n = 5 pet ghoup). "'1-hCG and othwt un.P_abeRed homone no.Ltion~ WQU injected inthaclvtdiac in 0.2 mL vehkkk which conni&ed 06 1% bovine .$ehum aLbumin in 0.9% ha&X. Ro& we@ nacti,$Lced2 hh~ ~&WL &j&tion, a time aZ which upAd~e orj 1251-hCG u~bnnhoum Xo be maximum (5,6). A dechetie -inihe htio 06 hU&oativily in the ovahy: bLood dwote-6 e66ecaXvc compe,ktion. Sf&ticicae nign.&$icance. in compatled ukth Group 1. NS = Not nigni&cant.
ApphoximcLt&ty 3 !~g] ludn injtckd
MAY 1983VOL. 27 NO. 5
517
CONTRACEPTION The data on ovulation induced by PMSG and hCG and the inhibitory activity of DG-hCG are shown in Tables II and III. In immature rats (Table II), PMSG treatment followed by hCG caused about a 2-fold increase in the number of ova However , administration of released into the oviduct (compare Grs. I and 4). 50 pg and 100 ug of DG-hCG after PMSG injection did not produce similar efthere was a tendency to decrease ovulation in the fects. On the other hand. 2, NS), with a statistically significant 50 ug DG-hCG treatment group (Gr. reduction in animals receiving 100 ug DG-hCG (Gr. 3). When these doses of DG-hCG were administered with hCG as in Groups 5 and 6, there was a significant reduction in ovulation suggesting inhibition of hCG action. However, there was no difference in the magnitude of reduction in these two groups (Grs. 5 and 6).
TABLE
II:
GA u U/J
OVULATION
T+UXLi%lCl~~ _____--_-___-_--_-___ KG
I
INHIBITION
(IU) DG-KG 0 0 0
BY DG-hCG
IN
IMMATURE FEMALE RATS
No. Ovu~tiing -_-_-_---_-_-
(LKJ) No.
/J&k cJILOU/J
0
g/10
50 100 0
315 4/5 6/6 5/6 717
* k + f ?r +
5.2 6.5 1.8 5.7 3.6 3.8
DG-hCG at 50 ug and 100 ug doses did not cause a significant reduction ovulation when injected on proestrus afternoon in adult cycling females (Table I I I). However, when the dose was increased to 200 ug per rat, there was 51% decrease in the number of ova released and this inhibition was statistically significant (P < 0.005 Grs. I and 4).
in
2 3 4 :,
518
25 25 25
50 100
21.1 13.6 5.7 48.1 31.0 30.0
2: t -t ;‘c tl9:
MAY 1983VOL. 27 NO. 5
CONTRACEPTION
TABLE
I I I:
EFFECT
OF DG-hCG ON 0VULAT:ON
IN MATURE CYCLING
RATS
DISCUSSION
The one-step chemical deglycosylation of hCG (1) produces a hormone product which has excellent receptor binding ability with virtually no biological effects. Such a molecule would be expected to have the ability to antagoThis has been demonstrated recently nize the action of the native hormone. in dispersed gonadal cells incubated ill vti’tu (2). The present data provide The metabolism of DG-hCG evidence which suggests its efficacy i~t wiv~?. which has virtually lost all of its terminal sialic acids in addition to other sugars. must be different from asialo hCG as seen by data in Table I. AS DG-hCG is effective in blocking the accumulation of lz51-hCG in the ovary under conditions in which asialo hCG is totally ineffective, it must mean that DG-hCG must remain in circulation long enough to bind to the receptors. Results of our preliminary studies indeed show that whatever DG-hCG that reaches the ovary remains bound to the receptors for a time much longer than that of lz51-hCG (6). The removal of sugar residues in addition to sialic acid are apparently responsible for the differences in the metabolic effects of DG-hCG and asialo hCG. Unlike asialo hCG which is rapidly eliminated by galactose-specific receptors in the liver (7), DG-hCG is not degraded by It should be mentioned this mechanism (M.R. Sairam, unpublished results). that DG-hCG is an antagonist of the action of hCG (2) while such activity is not intrinsic to asialo hCG. In addition to inhibiting the uptake of that DG-hCG can inhibit hormone-induced a 73% reduction in ova in PMSG-treated may be attributed to inhibition of the tary on the ovary. The same amount of decrease in ovulation induced by 25 IU not effective in mature rats but a dose
MAY 1983 VOL. 27 NO. 5
hCG, our data have also demonstrated The derivative caused ovulation. rats (Table II, Gr. 3),a result which action of LH released from the pituiDG-hCG (100 ug) caused about 37% Similar doses of DG-hCG were hCG. of 200 ug produced significant
519
CONTRACEPTION inhibition (51.4%, Gr. 4, Table Ill). Because of the difference in body weights between immature and mature rats, the latter apparently require more of the antagonist to cause inhibition. The results of ovulation inhibitory tests with DG-hCG are in agreement with the data of our previous study in which we used deglycosylated ovine LH as an antagonist we have shown that DG-hCG can successfully inhibit In other tests, (4). implantation and terminate an established pregnancy in the rat (8).
ACKNOWLEDGMENTS This investigation Geneva.
was
supported
in
part
by the
MRC of
Canada
and
the
WHO,
REFERENCES 1.
Biochemical, Manjunath, P. and Sairam M.R. cal properties of chemically deglycosylated J. Biol. Chem. 257: 7109-7115 (1982).
2.
Sairam, M.R. and Manjunath, chemically deglycosylated 258: 445-449 (1983) .
3.
Van Hall, E.V., Vaitukaitis, Immunological Ashwell, G. progressive desialylation.
4.
Sairam, hormonal
5.
Ashitaka, Y. and Koide, tropin with rat gonads.
S.S. Fertil
6.
Sairam, M.R., Russillo, of labeled gonadotropin Biol. Reprod. 26, rat.
B. and Manjunath, P. Ovarian and tissue uptake antagonists (LH and hCG) in the pseudopregnant Suppl. I, Abst. no. 233, 1982.
7.
The Ashwel I, G. and Morell, A.G. hepatic recognition and transport in Enzymol. 41: VT-128 (1974).
8.
Kato, K., termination antagonist.
520
Inhibition M.R. antagonist.
Hormonal antagonistic P. human choriogonadotropin.
J.L., Ross, and biological Endocrinology of LH-induced Contraception
113
ovulation in the rat 21: 651-657 (1980).
carbohydrates glycoproteins.
P. Inhibition of by a human chorionic (In
by a
of human chorionic gonado25: 177-184 (1974).
role of surface of circulating
(1983).
properties of J. Biol. Chem.
G.T., Hickman, J.W. and activity of hCG following 88: 456-464 (1971).
Interaction and Steril
M.R. and Manjunath, Sairam, of pregnancy in the rat Endocrinology
biological and immunologihuman choriogonadotropin.
in the Adv.
implantation gonadotropin
and
Press).
MAY 1983VOL. 27 NO. 5
INHIBITION
OF OVULATION
K.
IN
Kato
THE
and
RAT
M.R.
BY A hCG ANTAGONIST
Sairam
Reproduction Research Laboratory Clinical Research Institute of Montreal 110
Pine
Montreal,
Avenue
West
Quebec Canada
H2W lR7
ABSTRACT
Chemically shown
deglycosylated
to
have
pete with pregnant
1251-labeled rats. DG-hCG
PMSG-treated with
25
hormonal
hCG
its
antagonistic
hormonal
Submitted
MAY
hCG for binding at 100 ug dose
Administration reduced ovulation
was capable proestrus.
for for
publication publication
1983VOL.27NO.S
of
of
March
in
DG-hCG prior (P < 0.05).
in
ovulation indicate viwv
7,
1983
29,
1983
in
which were
the ovary ovulation
37%
by
activity
(DG-hCG) LM wtia
sites inhibited
of inhibiting These results
April
hCG
activity
rats.
IU
rats, DG-hCG afternoon of
Accepted
preparations antagonistic
to
have able
in by
been to
com-
pseudo73% in
and concurrent In mature cycling
when injected on the that DG-hCG can exert
rats.
515
CONTRACEPTION
INTRODUCTION Human choriogonadotropin (hCG) is a dimeric glycoprotein hormone whose However, the role chemical and biological properties are well known. of the bulky carbohydrate groups which comprise up to 35% of the molecule In a recent study, we showed that chemical has not been fully clarified. deglycosylation of hCG by anhydrous hydrogen fluoride treatment (HF) provided a hormone derivative with about 75% less carbohydrate (1). The modification which did not affect the polypeptide moiety of the hormone including its subunit structure had discordant effects on the biological properties of the hormone (l,Z), a feature with interesting potentials for elucidating its mechanism of action. The deglycosylated hCG (DG-hCG) which had an enhanced affinity for the hormone receptors in the testis and ovary and retained its immunological activity was virtually incapable of inducing hormone response in gonadal cells incubated ill utiu (l,Z). It was further demonstrated that the DG-hCG was an effective antagonist of the action of hCG or LH in W&W by inhibiting hormone-induced cyclic AMP accuSince DG-hCG virtually lacks sialic mulation and steroidogenesis (2). acid which is thought to be responsible for the relatively long half-life of hCG in circulation in rats (3), it was of interest to evaluate the antawe have investigated the gonistic activity in wivo. In the present study, effect of DG-hCG on ovulation using immature and mature rats.
MATERIALS
AND METHODS
hCG was purified from the commercially available crude powder and shown to have chemical and biological properties similar to a NIAMDDK preparation This was used for the preparation of deglycosylated hCG (DG-hCG) (CR 119). The dry hormone powder treated with hydrogen as described recently (1). fluoride for 60 mins at O°C was purified on concanavalin-A sepharose and sephadex G-100 (1). The DG-hCG was obtained as a stable lyophilized product which was stored at 4OC. Asialo hCG was prepared by treatment with immobilized neuraminidase (Sigma) and lyophilized.
Immature (40-45 Dawley CD rats were maintained 0500 h to 1900
g at 20 days of age) and adult (200-250 g) female Spraguewere purchased from Charles River Canada, Montreal. All rats on a 14-hr light - IO-hr dark lighting schedule (lights h) with food and wate.r ad LibLtum.
Inhibitian 06 upLake
06 lz51-hCG by VG-hCG
Immature female rats were made pseudopregnant by administration of 25 IU of PMSG on the 25th day followed by an injection of 25 IU of hCG 56-65 hrs later. The animals were used between days 6-8 after this treatment. The ability of unlabeled hCG, DG-hCG or asialo hCG to compete for binding sites for ‘251-hCG in the ovary was tested by administering them simultaneously. Two hrs after their intracardiac administration, the amount of radioactivity in the ovary and blood was determined and expressed as a ratio.
516
MAY
1983 VOL. 27 NO. 5
CONTRACEPTION Induction
o6 ovulation
Immature rats were maintained for 5 days and treated subcutaneously with The experimental schedule was identi25 Iu of PMsG (NIAMDDK, 1790 IU/mg). cal to that reported earl ier from our laboratory (4). Two days after PMSG, the DG-hCG or vehicle was administered subcutaneously with 50% of the dose distributed at 45 hrs and 48 hrs, respectively. This was followed by subcutaneous injection of 25 IU of hCG or vehicle at 48 hrs after PMSG. The animals were sacrificed under ether anesthesia 17-18 hrs after the last hormone injection and the oviducts were dissected. The number of ova released into the oviduct were examined and counted under the microscope as described previously (4). In this experiment PMSG and hCG solutions were prepared in saline. DG-hCG solution was prepared in 2% gelatin (Eastman Chemicals) with 0.9% NaCi and adjusted to pH 7.5 using IN NaOH. The effect of DG-hCG on ovulation in cycling rats was also tested by administering it on the afternoon of proestrus and examining for ova in the oviduct on the following day (see Table Ill). The
results
were
analyzed
for
statistical
significance
by Student’s
‘t’
test
RESULTS hCG and DG-hCG were effective in inhib As shown in Table I, both unlabeled ing the accumulation of 1251-labeled hCG in the pseudopregnant rat ovary. In marked contrast to this, up to 25 pg of asialo hCG was totally ineffect in competing with labeled hCG for binding sites in the ovary. TABLE
Ghoup
I:
Th&uinent '251-hCG p.Lw UvLeabeRed
I
Vehicle
2
hCG, 5 ug DG-hCG, 5.7 ug DG-hCG, 11.4 ug Asialo hCG, 25 ug
;
ive
INHIBITION OF UPTAKE OF lz51-hCG IN THE PSEUDOPREGNANT RAT BY UNLABELED HORMONES
----_-_-_-_-_
5
it-
Ratio
06 hU&Oativ~y
-in 100 mg ovw~q/lOO UP bLood Mean
120.0 .23.0 ill.0 19.0 101.6
* SEM
+ _+ * f *
6.0 0.6 2.3 3.6 9.8
P < 0.05 NS P < 0.05 NS
ng egtivafknt 06 lz51-hCG (npec&jicactivMy 60-90 i_~Ci/ into p~a~dopheynani htis (n = 5 pet ghoup). "'1-hCG and othwt un.P_abeRed homone no.Ltion~ WQU injected inthaclvtdiac in 0.2 mL vehkkk which conni&ed 06 1% bovine .$ehum aLbumin in 0.9% ha&X. Ro& we@ nacti,$Lced2 hh~ ~&WL &j&tion, a time aZ which upAd~e orj 1251-hCG u~bnnhoum Xo be maximum (5,6). A dechetie -inihe htio 06 hU&oativily in the ovahy: bLood dwote-6 e66ecaXvc compe,ktion. Sf&ticicae nign.&$icance. in compatled ukth Group 1. NS = Not nigni&cant.
ApphoximcLt&ty 3 !~g] ludn injtckd
MAY 1983VOL. 27 NO. 5
517
CONTRACEPTION The data on ovulation induced by PMSG and hCG and the inhibitory activity of DG-hCG are shown in Tables II and III. In immature rats (Table II), PMSG treatment followed by hCG caused about a 2-fold increase in the number of ova However , administration of released into the oviduct (compare Grs. I and 4). 50 pg and 100 ug of DG-hCG after PMSG injection did not produce similar efthere was a tendency to decrease ovulation in the fects. On the other hand. 2, NS), with a statistically significant 50 ug DG-hCG treatment group (Gr. reduction in animals receiving 100 ug DG-hCG (Gr. 3). When these doses of DG-hCG were administered with hCG as in Groups 5 and 6, there was a significant reduction in ovulation suggesting inhibition of hCG action. However, there was no difference in the magnitude of reduction in these two groups (Grs. 5 and 6).
TABLE
II:
GA u U/J
OVULATION
T+UXLi%lCl~~ _____--_-___-_--_-___ KG
I
INHIBITION
(IU) DG-KG 0 0 0
BY DG-hCG
IN
IMMATURE FEMALE RATS
No. Ovu~tiing -_-_-_---_-_-
(LKJ) No.
/J&k cJILOU/J
0
g/10
50 100 0
315 4/5 6/6 5/6 717
* k + f ?r +
5.2 6.5 1.8 5.7 3.6 3.8
DG-hCG at 50 ug and 100 ug doses did not cause a significant reduction ovulation when injected on proestrus afternoon in adult cycling females (Table I I I). However, when the dose was increased to 200 ug per rat, there was 51% decrease in the number of ova released and this inhibition was statistically significant (P < 0.005 Grs. I and 4).
in
2 3 4 :,
518
25 25 25
50 100
21.1 13.6 5.7 48.1 31.0 30.0
2: t -t ;‘c tl9:
MAY 1983VOL. 27 NO. 5
CONTRACEPTION
TABLE
I I I:
EFFECT
OF DG-hCG ON 0VULAT:ON
IN MATURE CYCLING
RATS
DISCUSSION
The one-step chemical deglycosylation of hCG (1) produces a hormone product which has excellent receptor binding ability with virtually no biological effects. Such a molecule would be expected to have the ability to antagoThis has been demonstrated recently nize the action of the native hormone. in dispersed gonadal cells incubated ill vti’tu (2). The present data provide The metabolism of DG-hCG evidence which suggests its efficacy i~t wiv~?. which has virtually lost all of its terminal sialic acids in addition to other sugars. must be different from asialo hCG as seen by data in Table I. AS DG-hCG is effective in blocking the accumulation of lz51-hCG in the ovary under conditions in which asialo hCG is totally ineffective, it must mean that DG-hCG must remain in circulation long enough to bind to the receptors. Results of our preliminary studies indeed show that whatever DG-hCG that reaches the ovary remains bound to the receptors for a time much longer than that of lz51-hCG (6). The removal of sugar residues in addition to sialic acid are apparently responsible for the differences in the metabolic effects of DG-hCG and asialo hCG. Unlike asialo hCG which is rapidly eliminated by galactose-specific receptors in the liver (7), DG-hCG is not degraded by It should be mentioned this mechanism (M.R. Sairam, unpublished results). that DG-hCG is an antagonist of the action of hCG (2) while such activity is not intrinsic to asialo hCG. In addition to inhibiting the uptake of that DG-hCG can inhibit hormone-induced a 73% reduction in ova in PMSG-treated may be attributed to inhibition of the tary on the ovary. The same amount of decrease in ovulation induced by 25 IU not effective in mature rats but a dose
MAY 1983 VOL. 27 NO. 5
hCG, our data have also demonstrated The derivative caused ovulation. rats (Table II, Gr. 3),a result which action of LH released from the pituiDG-hCG (100 ug) caused about 37% Similar doses of DG-hCG were hCG. of 200 ug produced significant
519
CONTRACEPTION inhibition (51.4%, Gr. 4, Table Ill). Because of the difference in body weights between immature and mature rats, the latter apparently require more of the antagonist to cause inhibition. The results of ovulation inhibitory tests with DG-hCG are in agreement with the data of our previous study in which we used deglycosylated ovine LH as an antagonist we have shown that DG-hCG can successfully inhibit In other tests, (4). implantation and terminate an established pregnancy in the rat (8).
ACKNOWLEDGMENTS This investigation Geneva.
was
supported
in
part
by the
MRC of
Canada
and
the
WHO,
REFERENCES 1.
Biochemical, Manjunath, P. and Sairam M.R. cal properties of chemically deglycosylated J. Biol. Chem. 257: 7109-7115 (1982).
2.
Sairam, M.R. and Manjunath, chemically deglycosylated 258: 445-449 (1983) .
3.
Van Hall, E.V., Vaitukaitis, Immunological Ashwell, G. progressive desialylation.
4.
Sairam, hormonal
5.
Ashitaka, Y. and Koide, tropin with rat gonads.
S.S. Fertil
6.
Sairam, M.R., Russillo, of labeled gonadotropin Biol. Reprod. 26, rat.
B. and Manjunath, P. Ovarian and tissue uptake antagonists (LH and hCG) in the pseudopregnant Suppl. I, Abst. no. 233, 1982.
7.
The Ashwel I, G. and Morell, A.G. hepatic recognition and transport in Enzymol. 41: VT-128 (1974).
8.
Kato, K., termination antagonist.
520
Inhibition M.R. antagonist.
Hormonal antagonistic P. human choriogonadotropin.
J.L., Ross, and biological Endocrinology of LH-induced Contraception
113
ovulation in the rat 21: 651-657 (1980).
carbohydrates glycoproteins.
P. Inhibition of by a human chorionic (In
by a
of human chorionic gonado25: 177-184 (1974).
role of surface of circulating
(1983).
properties of J. Biol. Chem.
G.T., Hickman, J.W. and activity of hCG following 88: 456-464 (1971).
Interaction and Steril
M.R. and Manjunath, Sairam, of pregnancy in the rat Endocrinology
biological and immunologihuman choriogonadotropin.
in the Adv.
implantation gonadotropin
and
Press).
MAY 1983VOL. 27 NO. 5