Species differences in the sensitivity to a GnRH antagonist

CONTRACEPTION

SPECIES DIFFERENCES IN THE SENSITIVITY TO A CnAH ANTAGONIST

P. Limontal,

C.W. Bardin, A. Ladishensk9ya, K. Sundaram and R.B. Thau

S. Pavlou,

Center for Biomedical Research, The Population Council, New York, NY 10021, USA ABSTRACT T$e effectf of the potent GnRH antagonist [AC-D-NAL(Z)‘, QF-D-Phe’, D-Trp D-Acg ]-CnRH (GnRH-A), on ovulation in mature rats and rabbits and on’serum LH and FSH levels in ovariectomized rats, rabbits and mice Dose-response studies showed that 1 pg (4 pg/kg) of were investigated. GnRH-A was sufficient to inhibit ovulation completely in cycling rats, while 500 pg (135 pg/kg) were required to inhibit mating induced ovulaTwo of the 3 rabbits which ovulated in spite tion in H of 11 rabbits. The mean LH peak of of the antagonist treatment had delayed LH surges. these 3 rabbits was significantly (p
1

Present Address! derbilt University 2

PL, Institut of Endocrinology, Milan, Medical Center Nashville, TN, USA.

to whom correspondence

Submitted Accepted

for publication for publication

should

SP,

Van-

be addressed

June 3, June 28,

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1985 1985

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INTRODUCTION The effects of CnRH agonists and antagonists on the pituitary and gonadal functions have been investigated in many animal species. These peptides are useful in the treatment of a variety of disorders associated with gonadal dysfunction. In addition, they provide a new approach for contraception in both men and women (1,2). Striking species differences have been reported in the sensitivity to the pituitary mediated and direct antitesticular effects of CnRH agonists (3-Y). Antagonists exhibit marked species differences in their antitesticular also effects (10). These findings in male animals prompted us to examine whether females also differ in the sensitivity to the antigonadal or antipituitary effects of CnRH antagonists. In female’rats the potency of antagonists is frequently tested by evaluating the dose needed for suppresbecause of the differences in the sion of ovulation (11). However, cycle and hormonal regulation of induction of ovulation, a reproductive comparison between species that ovulate spontaneously (i.e. rats and and induced ovulators (i.e. rabbits) is problematic. mice) Therefore, in addition to comparing suppression of ovulation in these species, we thought it pertinent to examine the effects of a single dose of CnRH-A on serum gonadotropin concentrations in ovariectomized animals. Ovariectomy was performed in order to create a hormonal state that can be compared more objectively in its response to GnRH antagonists. The results of this study showed that the antigonadal as well as antipituitary effects of the antagonist vary significantly among female rats, rabbits and mice and that the rat was the most sensitive animal. MATERIALS AND METHODS Hormone

preparations

The GnRH antagonist, [AC-D-NAL(2)1, 4FD-Phe’, D-Trp3, D-Arg’]-GnRH dissolved in normal saline or 40% propylene glycol in (GnRH-A), was saline and injected subcutaneously (6.c.) or given orally (p.0.). Animals

and Treatment

Female Sprague-Dawley rats (250-260g BW) were purchased from Taconic Inc.,Germantown, NY; NCS mice (Swiss, 30-358 BW) from the breedFarms, New Zealand ing colony at Rockefeller University, New York, NY; and white rabbits (2.5-3.9 kg BW) from Dutchland Laboratories, Denver, PA. All animals were housed under standard conditions and fed a commercial Blood samples were obtained from the median ear artery of rabbits diet. and from the trunk of rats and mice following decapitation. with at least two consecutive 4-day Experiment I: Cycling rats cycles were injected S.C. with GnRH-A at 12 noon of proestrus and killed the following morning. Controls received vehicle (40% propylene glycol The oviducts were dissected out and compressed firmly in saline) only. The presence of ova was examined under between 2 microscopic slides. Immature rats were treated as described by Sundaram & the microscope.

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a_l.

injected except that 5 IU of PMSG was was injected on day 30 and the presence following morning as described above.

(12),

GnRH-A

the

Mature female rabbits were injected immediately before mating. The h, or measured. Ovulation and pregnancy were levels. Pregnancy length and number of Experiment II: The examined in rats, mice, least 4 weeks. Time of 10 or 100 (0.1, 1, injected S.C. at O-time. Hormone

of

on day 28 of age. ova was determined on

0.5 S.C. with GnRH-A at 4.0 h, ovulatory LH-surge in plasma was confirmed by plasma progesterone fetuses were recorded.

suppression of serum gonadotropin levels was and rabbits which had been ovariectomized for at blood sampling is shown in Figure 3. GnRH-A pg/kg BW in 0.2 ml normal saline) or vehicle was

Am

Serum LH and PSH concentrations were measured by using RIA kits prepared by Dr. A.F. Parlow and distributed by the NIAMDD hormone distribution program. Serum LH levels in rabbits are expressed in ng/ml of LH-AFP-559-B. The limit of detection was 0.1 ng/ml. Intra- and interassay coefficients of variation were 7.9 and 12.98, respectively. Rat LH and FSH were expressed in terms of the respective rat RP-2-standards. Intraand interassay coefficients of variation were 7.5 and 9.4%, respectively, for LH and 4.0 and 7.8% for PSH. The limits of detection were 0.2 ng/ml for LH and 0.4 ng/ml for FSH. Mouse LH and FSH were expressed as rat RP-2-standard-equivalents. Antirat antiserum and iodinated rat hormones were used in the mouse RIA.

Serum progesterone was determined according to the method of Thorneycroft and Stone (13). The antiserum was prepared in rabbits, using Cross-reactivity llc(-succinylhydroxyprogesterone-BSA. was 0.72 for 2Oo(-hydroxy-4-pregnen-3-one, 0.9% for for 17<-hydroxyprogesterone and 0.28 for 3a-hydroxy-5-pregnen-20-one. The limit of detection was 5.4 Intraand interassay coefficients of variation were 8.6 and 17.78, Pg. respectively (14). Statistical

Analysis

Statistical analysis was performed using the BMDP statistical packHormone age (15). levels were subjected to analysis of variance, differences between means were analyzed according to the Bonferroni test for multiple comparisons. The Chi-square test was used for statistical analysis of ovulation inhibition. RESULTS Experiment I: The dose response studies in cycling rats showed that 1 pg (4 ug/kg) of GnRH-A S.C. was sufficient to inhibit ovulation (Table I). The same dose was also effective S.C. in PMSG-treated immature rats, while 5 mg (1.35 mg/kg) were needed to inhibit ovulation in 8 of 10 rats following oral administration.

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Table

I: Ovulation Inhibition in Cycling Immature Rats with CnRH-A

No. Rats Ovulating/ No. Rats Treated

Dose

Group

WRat

Cycling

Rats,

6.c.l

Immature Rats,

Immature Rats,

s.~.~

p.o.2

1 . 21njected S.C. at Injected with 5 examined on day Chi-square-Test:

Table Time of Administration’

Oh

-0.5h

II:

0 0.2 1 5

?/9a 7/1g 0/9b O/9

0 1

::$

0 200 ZODO 5000

7/10e 9/10 10/1p 2/10

12 noon of ptoestrus and examined at 9 A.M. of estrus, Ill PMSCon day 28, with CnRH-A, S.C. or ~.a., on day 30 and 31. a/b! p < 0.001, c/d: p < 0.05, e/f: p C 0.025.

Inhibition

of Mating-Induced

Dose pg/Rabbi t

in Rabbits No. Fetuses/ Rabbit

313 4/5

N.D.

N.U

31.1 L 0.6

7.8 + 0.B

500

12/14” 3/b 4;s 3/11*

32.0 + 0

8.0 t 0

100 500

3/4 3/4

32.6 + 0.4

7.B + 1.0

100 500 0 100

No. Rabbit Ovulating/ No. Treated

Ovulation Gestation Time Pays 1

5

-4.Oh

and

1 Mating at O-time Whi-squareTest! p < 0.005 N.D. - Not determined

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In rabbits, GnRH-A was less effective in inhibiting (mating induced) ovulation. Even the highest dose tested (500 pg per rabbit, 135 pg/kg 4h or immediately before mating BW) had no effect when administered (Table II). However, when treatment was given 0.5h before mating B of 11 rabbits were anovulatory. Two of the three rabbits which ovulated following GnRH-A administration had delayed LH surges which peaked at 4h as compared to 2h for vehicle-treated controls (Fig. 1). In addition the mean LH peak (ug/ml serum) of the 3 rabbits was significantly lower (p < 0.001) than that of controls (6.0 + 1.4, N=3 vs 18.4 + 3.3 &rum prog&teroneN=Z Z”$$steTf~e TZrlX induced increase in throughout pregnancy were comparable to those in normal pregnant rabbits (Fig. 2). The duration of gestation (32.0 + 0 days, N=3, vs. 31.1 t 0.6, N=B, for controls) and the average number of fetuses (8.0 + 0 vs. 7.8 + 0.8 for controls) were also unaffected by GnRH-A treatment. Experiment II: Serum LH levels of castrated rats were suppressed in a dose-related manner following the S.C. administration of GnRH-A (Fig. 3). The highest dose (10 pg/kg) induced a significant (p ( 0.01) decline in LH concentration lh after administration. The lowest LH level was reached at 2h and persisted through 6h. By 24h LH levels had returned to pre-injection values. Serum FSH showed a similar but smaller response. In rabbits the initial decline in serum LH levels following the administration of 10 pg/kg GnRH-A was similar to that found in rats (Fig. 3). However, by 4h LH levels had started to increase and had risen to approximately 50% of that of controls by 6h. By 24h they were not statistically different from controls. In rabbits receiving 100 ug/kg GnRH-A the serum LH levels remained suppressed for up to 24 h. By 48 h the LH levels returned to normal. Neither dose caused a significant decrease in serum FSH levels. This lack of FSH response was also observed in castrated mice (Fig. 3). Moreover, in mice the effect of the GnRH-A was smaller and of shorter duration than in rabbits. Treatment with 10 pg/kg caused a decrease in serum LH to approximately 501 of control levels by 0.5 h. LH concentrations began to rise by 1 h and returned to normal post-castration values by 4h. Even at 100 pg/kg, serum LH levels were suppressed for only for 2 h. DISCUSSION These studies extend the observations previously made in males and show that significant species differences in the sensitivity to GnRH antagonists exist also among females. Ovulation could be inhibited in rats using 4 pg/kg BW of GnRH-A, while 34 times this dose only partially suppressed ovulation in rabbits. Rabbits are induced ovulators and the time of antagonist administration seems to be critical. Reel et al. (16) administered less potent GnRH antagonists to rabbits immedr ately before and after mating and failed to inhibit ovulation, although these antagonists were capable of blocking spontaneous ovulation in rats and hamsters. In our experiments, even the highest dose used (135 pg/kg BW) was ineffective when administered immediately or 4h before mating, while only 3 of 11 rabbits ovulated when the GnRH-A was injected 0.5h before mating. Even in the 3 rabbits which ovulated and subsequently

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0246 HOURS

AFTER

MATING

Fig. 1. Plasma LH levels (mean*SE) in rabbits following mating at time treated with GnRH-A Animals shown by the open symbols were a. (500pg/rabbit) and those by the closed symbols with vehicle (N=12) S.C. ovulatory rabbits: open triangles, 0.5h before mating. CnRH-A-treated, *p~O.OOl. N=3; anovulatory rabbits! open circles, N=8

E

0

2

4

HOURS

6

1

7

15

23

31

DAYS

Fig. 2. Plasma progesterqne levels (mean f. SE) in rabbits at various times after mating. Animals were treated with CnRH-A or vehicle s.c., 0.5 h beEore mating. Vehicle Controls: closed circles, (N=6) ; GnRH-Atreated rabbits that ovulated and became pregnant, closed triangles (N=3); CnRH-A-treated rabbits that presumably did not ovulate, closed diamonds, N=S.

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Fig. 3: Plasma LH and FSH levele (mean + SE, N=4 to 8) in ovariectomized animals at different times following treatment with vehicle or CnRH-A. Left Panel: Rats. Closed circles: Vehicle control; closed diamonds: 0.1 pg/kg; open diaopen circles: 10 pg/kg. Significantly different from monds: 1 w/kg: control: a=p<0.05; b=pcO.Ol; c=p
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became Pregnant, the LH peak was significantly diminished and in 2 of them it was also delayed. A similar delay was reported by Phelps & a_l. (17) who used 4 mg/kg of another antagonist. The fact that the cour6e of pregnancy was normal (serum progesterone levels, number of fetuses and length of gestation) following significantly reduced LH surges, suggests that only a fraction of the LH released in response to copulation is necessary to induce ovulation and the formation of functionial corpora lutea. The “surplus LH” may constitute a safety mechanism to insure reproductive success. In the B non-ovulating rabbits the suppression of serum LH levels by GnRH-A was complete. In spite

of the differences

in the hormonal

the regulation of reproductive make a comparison between rats clearly show that the rabbit effects of GnRH-A than the rat.

mechanisms involved in cycles and induction of ovulation which and rabbits difficult, results our is less sensitive to the antiovulatory

The differences between rats and rabbits may be due to differences in pituitary receptor binding. Although antagonists like agonists bind to rat gonads, no direct inhibitory action by antagonists has been observed (18‘19). We have shown that male rats are more sensitive to pituitary-mediated effects of CnRH-A Rats are also more sensitive to direct gonadal than rabbits (10). effects of CnRH agonists than mice (3,7,20) and monkeys (4-6,21,22). This is consistent with the finding that mice and monkeys show few or no Recently Koos & al_. (24) reported gonadal CnRH binding sites (3,23). significantly reduced sensitivity in vitro to ovulation induction with a superactive agonist for the rabbit ovary as compared to the rat ovary. In the intact animal, ovulation is regulated via pituitary GnRH receptors and the inhibitory actions of GnRH antagonists are most likely Our observations that the affinity of exerted via the same receptors. GnRfl-A to pituitary receptors is 4-to 5-fold higher in rats than in rabbits (25) suggests that differences in receptor binding play a role in the observed species differences in the sensitivity to GnRH-A. However, in GnRH-A- metabolism and clearance may in part species differences explain our findings and need to be explored. Differences in GnRH-A binding by the pituitary are also a likely cause for our findings in castrated females. Suppression of LH release following GnRH-A treatment clearly indicated that the rabbit and the mouse pituitary are less sensitive to GnRH-A effects than the rat. The the rat and a in extent and duration of LH suppression was highest reduction of PSH levels following antagonist treatment was observed only in the

rat,

even

at

the

highest

doses

used.

GnRH-A treatment at doses which do The lack of PSH response to However, it is in rabbits and mice was surprising. affect LH release rats and supconsistent with the diminished PSH response observed in ports previous suggestions that PSH secretion in vivo may be less dependent on acute GnRH secretion than LH (26). In summary, the results studied, similar to species tive animal in its response GnRH antagonist. Because

of the present study the male, the female to the antipituitary of

the

species

show that among the rat is the most sensieffects of a Potent

differences,

findings

from

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animal models made regarding

require their

careful possible

interpretation before predictions effects in the human.

can

be

ACKNOWLEDGEMENTS This work was funded in part by NIH Grant HD 13541 and was undertaken as part of the contraceptive development program sponsored and coordinated by the International Committee for Contraception Research of The Population Council, Inc., New York, NY. The financial support provided by the International Development Research Centre of Canada, the U.S. Agency for International Development (Grant AID/pha 1116), the Ford and the George J. Hecht Fund is Foundation, the Rockefeller Foundation, gratefully acknowledged. The content of this report does not necesCnRH analogs sarily reflect the policy of any of the funding sources. were kindly supplied by Drs. J. Rivier and W. Vale of the Peptide BiolCalifornia. We thank Linda ogy Laboratory,Salk Institute, La Jolla, McKeiver and Susan Richman for their skillful secretarial assistance. REFERENCES 1.

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