0021-972X/90/7004-1213$02.00/0 Journal of Clinical Endocrinology and Metabolism Copyright© 1990 by The Endocrine Society
Vol. 70, No. 4 Printed in U.S.A.
The Effect of Intraluteal Infusion of Deglycosylated Human Chorionic Gonadotropin on the Corpus Luteum in Rhesus Monkeys* PHILLIP E. PATTON, RICHARD L. STOUFFER, AND MARY B. ZELINSKI-WOOTEN Department of Obstetrics and Gynecology, Oregon Health Sciences Center (P.E.P.), Portland, Oregon 97201; and the Division of Reproductive Biology and Behavior, Oregon Regional Primate Research Center (R.L.S., M.B.Z.), Beaverton, Oregon 97006
ABSTRACT. Removal of the carbohydrates from hCG results in an antagonist (degly-hCG) that competitively inhibits hCG/ LH-stimulated adenylate cyclase in macaque luteal tissue in vitro, but its effect in vivo is controversial. To examine the effect of degly-hCG on the lifespan and steroidogenic activity of the primate corpus luteum, the antagonist was administered to female rhesus monkeys (n = 7) beginning at the midluteal phase of the menstrual cycle. In a control cycle the saline vehicle was infused via an osmotic minipump directly into the corpus luteum. In a subsequent cycle, one of three dose rates of degly-hCG (0.001, 0.009, and 0.09 nmol/h) was infused into the corpus luteum. Pump implantation and infusion began 5-9 days after the midcycle LH surge and continued for 7 days. Peripheral
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venous blood was collected daily from day 8 of the cycle until menses, and serum progesterone levels were determined by RIA. Progesterone levels and patterns were similar in animals that received either the saline vehicle or degly-hCG, and the length of the luteal phase in monkeys receiving any dose of degly-hCG (16.4 ± 0.5 days) was not different from that in animals receiving a control infusion (16.1 ± 0.9 days). In a corollary study, an intraluteal infusion of degly-hCG (0.009 nmol/h) in the midluteal phase did not prevent stimulation of progesterone levels after im injection of hCG (15 IU/day for 5 days). We conclude that whereas degly-hCG is a useful tool to examine gonadotropin action in vitro, it is not a potent gonadotropin antagonist in vivo. (J Clin Endocrinol Metab 70: 1213-1218, 1990)
CG AND LH contain a series of complex carbohydrate side-chains attached to amino acid residues in both the a- and /?-subunits. Because removal of the carbohydrate decreases the in vitro action of gonadotropic hormones, the oligosaccharide moieties are considered obligatory for the full expression of biological activity. Chemical deglycosylation of purified hCG results in a stable compound (degly-hCG) with 85% of its carbohydrate residues removed, which acts as an effective antagonist of gonadotropin action in rodent luteal tissue in vitro (1). Moreover, degly-hCG inhibits LH/ CG-stimulated cAMP and steroid production in primate luteal tissue in vitro (2, 3). Despite evidence that degly-hCG acts as an effective gonadotropin antagonist in vitro, few studies have assessed the in vivo effects of degly-hCG on gonadotropindependent tissues. In the rodent, the administration of
degly-hCG apparently interferes with gonadotropindependent ovulation, implantation, and early pregnancy (4, 5). Alternatively, the systemic administration of degly-hCG in the primate has not been shown to inhibit the response to endogenous gonadotropins in LH/CGdependent tissues (6, 7). Nevertheless, the short term infusion (6) and the rapid metabolic clearance of the compound could have limited the antagonism of deglyhCG on gonadotropin tissues. Recently, methods to deliver substances directly to the corpus luteum were developed (8, 9), which circumvents the problems associated with systemic administration. To examine further the in vivo action of degly-hCG on primate luteal function, the antagonist was infused chronically and directly into the corpus luteum of rhesus monkeys beginning at the midluteal phase of the menstrual cycle.
Received August 17,1989. Address requests for reprints to: Phillip E. Patton, M.D., Department of Obstetrics and Gynecology, L-466, Oregon Health Sciences University, 3181 SW Sam Jackson Park Road, Portland, Oregon 97201. * This work was supported by the Medical Research Foundation of Oregon (tp P.E.P.), NIH Grant HD-20869 (to R.L.S.), and NIH Grants HD-18185 and RR-00163. Oregon Regional Primate Research Center Publication 1697.
Materials and Methods degly-hCG
Degly-hCG prepared by chemical treatment of purified hCG was a gift from Dr. Robert Ryan (Mayo Clinic, Rochester, MN). The preparation and in vitro characterization of the compound 1213
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have been described in detail previously (1). No demonstrable agonist activity was apparent when cAMP production was measured in rat ovarian membranes exposed to doses of deglyhCG up to 104 pmol (10). Experimental design The housing and general care of rhesus monkeys at the Oregon Regional Primate Research Center were described previously (11). Monkeys were checked daily for menses, and menstrual records were maintained. Adult females exhibiting normal menstrual cycles of approximately 28 days were used in the study. Saphenous blood samples were drawn daily between 08000830 h from day 8 of the menstrual cycle until menses or pump removal, whichever occurred later. Serum estradiol and progesterone concentrations were measured in the Core Hormone Assay Laboratory at the Oregon Regional Primate Research Center using previously described RIA methodology (12, 13). The intra- and interassay variations were 8.3% and 9.8% for estradiol and 10.7% and 16.5% for progesterone, respectively. An osmotic minipump (Alzet, model 2ML1, Alza Corp., Palo Alto, CA), which delivers between 9.5-10.5 juL/h continuously for 7 days, was implanted sc in the flank of female rhesus monkeys during the midluteal phase (5-9 days after the LH surge) of the menstrual cycle. The day of the LH surge was estimated to occur within the same 24-h interval of the peak preovulatory estrogen level (14). The mean day of pump implantation did not differ significantly among control and treatment groups. The description of the anesthesia and surgical procedures used for pump implantation has been detailed previously (9). The osmotic minipump was secured sc, and a PV4 catheter connected to the pump and ending in a 21-gauge needle was inserted directly into the corpus luteum. Each osmotic minipump was removed 7 days after placement, and the pump reservoirs were inspected to confirm delivery of the infusate. The needle was removed from the ovary during a subsequent laparotomy, generally within 3 months. The study design was such that each animal (n = 7) received an intraluteal infusion of vehicle during a control cycle before a degly-hCG treatment cycle. The saline vehicle consisted of 0.15 M NaCl, pH 7.4, and 0.1% BSA (wt/vol; Sigma Chemical Co., St. Louis, MO). Only animals that exhibited a normal progesterone profile both before and after pump placement in the luteal phase were tested in subsequent treatment cycles. A normal progesterone profile was defined as progesterone values within the 95% confidence interval of progesterone values from comparable days of the menstrual cycle in untreated animals (n = 18) from our colony (10). After the control cycle, one of three dose levels of degly-hCG [0.001 nmol/h (n = 2), 0.009 nmol/h (n = 3), and 0.09 nmol/h (n = 3)] was infused into the corpus luteum during the treatment cycle. In the calculations, the following estimated mol wt were used: 40K for hCG and 31.6K for degly-hCG. The intraluteal dose of degly-hCG necessary to act as a competitive antagonist of LH was estimated from peripheral LH levels and blood flow to the macaque ovary at the midluteal phase of the cycle (15). In a corollary study, either the saline vehicle (n = 1) or degly-hCG (0.009 nmol/h; n = 1) was infused into the
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corpus luteum during the midluteal phase as described previously. Two days after pump implantation, intact hCG (Ayerst APL, Rouses Point, NY; 15 IU/day) was given im for a total of 5 days in each animal. In a final study, intact hCG (15 IU/day) was infused into the corpus luteum of two monkeys as previously described (9). Adenylate cyclose assay Luteinized ovaries of superovulated rats were pooled, homogenized, and centrifuged to obtain a membrane particulate fraction, according to methods previously described (2). The activity of adenylate cyclase was examined using established procedures (2), and activity was assessed in the presence and absence (basal activity) of maximally stimulating doses of GTP (2.5 nmol) and hCG (12.5 pmol; CR123). In addition, freshly prepared degly-hCG (12.5 pmol) and degly-hCG recovered from a minipump after 7 days in vivo (47.5 pmol) were incubated with rat luteal membranes in the absence and presence of 12.5 pmol hCG. The activity evoked by each addition was assayed in triplicate, and the results were expressed as the mean ± SEM. Data were expressed as moles of cAMP produced per min/mg protein. Statistical analysis Daily progesterone levels from animals that received the saline vehicle infusion or intraluteal degly-hCG at the three dose levels tested were analyzed using two-way analysis of variance with repeated measures over time. Luteal phase lengths from the same groups were analyzed by one-way analysis of variance. Differences were considered significant at a level of P< 0.05.
Results Progesterone patterns in monkeys receiving an infusion of the saline vehicle beginning at the midluteal phase of the cycle are shown in Fig. 1. As noted previously (10), serum progesterone levels were lower in the sample obtained 24 h after surgery, but levels in the postpump implantation interval remained in the normal range compared to those in untreated animals. In one of the seven monkeys, progesterone values dropped precipitously, followed by menses on the third day after pump implantation and infusion with the saline vehicle (data not shown). When tested in a subsequent control cycle (data included in Fig. 1), a normal progesterone profile and luteal phase length were noted in this animal. The effect of an intraluteal infusion of degly-hCG at the highest dose tested (0.09 nmol/h) on progesterone profiles is illustrated in Fig. 2. Progesterone profiles obtained with lower infusion rates were similar; therefore, a single representative figure (Fig. 2) is included. As in the saline vehicle-treated monkeys, progesterone levels declined at 24 h, but remained within the normal range during the infusion interval. At all three dose levels tested there were no differences in progesterone levels in
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Days Relative to Pump Implantation FIG. 1. Patterns and levels (mean ± SEM; n = 7) of progesterone in monkeys receiving intraluteal infusions of 0.9% normal saline and 0.1% BSA. Data are normalized to the day of pump implantation (day 0). The horizontal bar on the x-axis indicates the treatment interval. The shaded area represents the 95% confidence interval for serum progesterone levels during this time in the menstrual cycle based on values from untreated animals (n = 18) in our colony. To convert the results to SI units, multiply nanograms per mL P by 3.18 nmol/L.
animals treated with degly-hCG vs. the saline vehicle. As summarized in Table 1, the interval from pump implantation until menses or the length of the luteal phase was not significantly different between animals that received either the saline vehicle or any of the three dose levels of degly-hCG tested. Figure 3 illustrates the progesterone profiles from two monkeys that received either an intraluteal infusion of saline vehicle or degly-hCG (0.009 nmol/h). Both animals received exogenous hCG (15 IU/day, im) for 5 consecutive days, beginning 2 days after pump implantation. Each animal exhibited a similar agonist response to hCG, as reflected by an increase in circulating levels of progesterone within 24 h to 3-fold above the normal range, which was sustained for 4-5 days. The interval from pump implantation until menses was 8 days for each animal. Similarly, the infusion of antagonist for 48 h before the injection of hCG had no effect on the length of the luteal phase compared to that in the saline vehicle-
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Days Relative to Pump Implantation FIG. 2. Patterns and levels (mean ± SEM) of progesterone in monkeys receiving an intraluteal infusion of degly-hCG (0.09 nmol/h; n = 3). Refer to Fig. 1 for details. TABLE 1. Interval between pump placement and menses and length of the luteal phase Intraluteal infusate Saline vehicle (n = 7) Degly-hCG 0.001 nmol/h (n = 2) 0.009 nmol/h (n = 3) 0.09 nmol/h (n = 3)
Interval to menses
Luteal phase length0
9.7 ± 1.76
16.1 ± 0.9*
11.0 8.0 ± 1.0 10.3 ± 2.1
17.0 15.3 ± 0.6 17.0 ± 2.0
Values are the mean (days) ± SEM. ° Day of LH surge to onset of menses. b There were no differences in the interval from pump placement to mense or length of the luteal phase among treatment groups.
treated animal (15 and 16 days, respectively). The effect of an intraluteal infusion of intact hCG on progesterone profiles is depicted in Fig. 4. In contrast to infusions of saline vehicle or degly-hCG (Figs. 1 and 2), there was no apparent drop in progesterone levels 24 h after pump implantation. Using a dose of hCG (15 IU/ day) comparable to the im dose in the previous study,
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Days Relative to Pump Implantation Days Relative to Pump Implantation FIG. 3. Progesterone levels in monkeys receiving an intraluteal infusion of saline (O-O) or degly-hCG (•- -•; 0.009 nmol/h). Both animals received hCG (15 IU/day) for 5 consecutive days, beginning 2 days after pump placement (hCG, 15 IU/day in box).
progesterone levels increased within 24 h of infusion and remained elevated above the normal range for 4-5 days before declining. Table 2 summarizes adenylate cyclase activity in rat luteal membranes in the absence and presence of intact hCG and degly-hCG recovered 7 days after pump implantation in vivo. Both freshly prepared degly-hCG and degly-hCG recovered from the minipump failed to stimulate cAMP production and inhibited hCG-stimulated cAMP production by rat luteal homogenates.
Discussion The primate corpus luteum in the nonfertile cycle appears to be dependent upon pulsatile exposure to pituitary LH. Investigators report an early demise of the corpus luteum when anti-LH antiserum, anti-CG antiserum, or GnRH antagonists are administered to women or monkeys during the luteal phase (16-18). Similarly, using the hypothalamic clamped monkey model with bilateral lesions in the arcuate region of the hypothala-
FIG. 4. Progesterone levels in two monkeys (D and • ) receiving intraluteal infusions of hCG (15 IU/day). Refer to Fig. 1 for details. TABLE 2. Adenylate cyclase activity of rat luteal membranes in the absence and presence of intact hCG, degly-hCG, and degly-hCG recovered 7 days after pump implantation in vivo
Degly-hCG 0 12.5 pmol (fresh) 47.5 pmol (postpump)
cAMP production (pmol/min protein) Control0
12.5 pmol hCG
10.2 ± 0.3 11.6 ± 0.8 9.0 ± 0.5
31.6 ± 2.2 20.4 ± 1.66 16.2 ± 2.2*
Values are the mean ± SEM of triplicate determinations. GTP (2.5 nmol) was present in control and hCG treatment groups (basal activity, 5.8 ± 0.7). b Both freshly prepared degly-hCG and degly-hCG recovered from an osmotic minipump after 7 days in vivo inhibited hCG-stimulated cAMP production by rat luteal homogenates. 0
mus, the withdrawal of GnRH-driven gonadotropin support in the midluteal phase of the menstrual cycle results in a prompt reduction of progesterone and menses (19). Although these studies support a gonadotropin-dependent corpus luteum, there is concern that the substances used or the model employed may include an effect other than an anti-LH action, which influences the function
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or lifespan of the corpus luteum. Furthermore, in view of studies that propose a gonadotropin-independent corpus luteum (20, 21), the mechanisms regulating corpus luteum function remain unresolved. We employed a more direct method of testing the dependence of the corpus luteum on gonadotropins in vivo by locally infusing a substance (degly-hCG) that has been shown to be a gonadotropin antagonist in primate tissues in vitro (2, 3). We hypothesized that the administration of degly-hCG would occupy the luteal cell LH receptor, prevent binding of circulating LH, diminish progesterone secretion, and induce early menses. Nevertheless, infusion of the LH/hCG antagonist into the corpus luteum beginning in the midluteal phase of the menstrual cycle resulted in no change in progesterone levels or luteal phase length compared to those in a control group that received an infusion of saline vehicle. At all dose levels tested there was no evidence that deglyhCG acted as an effective gonadotropin antagonist in macaque luteal tissue in vivo. The failure of degly-hCG to induce early menses is not likely to be due to a subtherapeutic dose, inadequate delivery or distribution of the compound to the corpus luteum, or instability of the compound. First, peripheral serum LH levels (mean ± SEM) measured by bioassay in the rhesus monkey approximate 8.3 ±3.5 ng/mL (using NIH RP-1 standard) in the luteal phase of the menstrual cycle (10). Assuming an ovarian blood supply of approximately 100 mL/h in the luteal phase (16), a 0.09 nmol/ h dose of degly-hCG should deliver approximately a 3fold molar excess of degly-hCG compared to LH to the luteal cell. At the highest concentration used (0.09 nmol/ h), 2.16 nmol degly-hCG would be directly delivered to the corpus luteum per day. Second, it is also unlikely that degly-hCG failed to act as an antagonist because of impaired delivery or poor distribution to the corpus luteum. In two monkeys, intraluteal infusion of a low dose of hCG resulted in a prompt increase in progesterone levels above values in control cycles. Moreover, the generation of antibodies to CG-like molecules by two of three animals (data not shown) implies that the compound was infused in significant quantities. Furthermore, it is doubtful that our results can be explained by instability of the degly-hCG over the interval of infusion. Degly-hCG recovered from the minipump after 7 days in vivo inhibited hCG-stimulated adenylate cyclase activity in rat ovarian membrane homogenates in vitro. The results of our preliminary studies indicated that degly-hCG was not a potent gonadotropin antagonist in vivo. However, the possibility that the corpus luteum is independent of LH support could not be excluded. Therefore, we examined the response of the corpus luteum to exogenous hCG after preexposure to degly-hCG. Previous studies indicated that the incubation of ovarian
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membranes with increasing concentrations of degly-hCG progressively diminishes the adenylate cyclase response to hCG in vitro (2). Nevertheless, the infusion of deglyhCG at a dose of 0.009 nmol/h for 48 h did not inhibit the agonist action of low dose hCG (15 IU/day, im) on corpus luteum function in vivo. Progesterone profiles and luteal phase lengths after exogenous gonadotropin treatment were indistinguishable in monkeys pretreated with degly-hCG or saline vehicle. In contrast to in vitro studies, our results indicate that the infusion of degly-hCG has no effect on basal (LH-supported) or hCG-stimulated corpus luteum function during the menstrual cycle. We conclude that whereas degly-hCG is a useful tool to examine gonadotropin action in vitro, it is not a potent gonadotropin antagonist in primate luteal tissue in vivo. Additional observations support the finding that deglyhCG is not an effective LH/hCG antagonist in primate species in vivo. In women, the peripheral infusion of doses as high as 0.25 nmol/kg-day degly-hCG during the midluteal phase of the menstrual cycle had no effect on daily progesterone levels or the duration of the luteal phase (6). Moreover, a single iv injection of degly-hCG (0.32 ± 0.02 nmol/kg) into the male macaque actually led to an increase in testosterone levels compared to the response to an equimolar dose of hCG, suggesting an agonist effect on Ley dig cell steroidogenesis (7). Therefore, it is possible in the current study that residual agonistic activity of degly-hCG maintains the corpus luteum despite antagonism of circulating gonadotropins, as observed in the male macaque in vivo and gonadotropin-dependent tissues in vitro (3, 22). In summary, the infusion of degly-hCG directly into the corpus luteum had no effect on the patterns of circulating progesterone or the length of the luteal phase in rhesus monkeys. Thusfar, the data obtained after bolus injection, short term peripheral infusion, or chronic intraluteal infusion provide no substantial evidence of antagonism of gonadotropin-stimulated action by deglyhCG in primates in vivo. It is doubtful that degly-hCG will prove to be an effective luteolytic agent in primates in the future. Acknowledgments We would like to thank Dr. Robert Ryan for the generous gift of degly-hCG, and Dr. David Hess and his staff for performing steroid assays. Special thanks are due to Dr. Ted Molskness for technical advice and support, and Elizabeth Cook for her secretarial services.
References 1. Keutmann HT, Mcllroy PJ, Bergert ER, Ryan RJ. Chemically deglycosylated human chorionic gonadotropin subunits: characterization and biological properties. Biochemistry. 1983;22:3067-72. 2. Eyster KM, Stouffer RL. Adenylate cyclase in the corpus luteum of the rhesus monkey. Sensitivity to nucleotide, gonadotropins, catecholamines, and nonhormanal activators Endocrinology.
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1985;116:1552-8. 3. Richardson MC, Masson GM, Sairam MR. Inhibitory action of chemically deglycosylated human chorionic gonadotropin on hormone-induced steroid production by dispersed cells from human corpus luteum. J Endocrinol. 1984;101:327-32. 4. Kato K, Sairam MR, Manjunath P. Inhibition of implantation and termination of pregnancy in the rat by a human chorionic gonadotropin antagonist. Endocrinology. 1983; 113:195-99. 5. Kato K, Sairam MR. Inhibition of ovulation in the rat by an hCG antagonist. Contraception. 1983;27:515-20. 6. Patton PE, Calvo FO, Fujimoto VY, Bergert ER, Kempers RD, Ryan RJ. The effect of deglycosylated human chorionic gonadotropin on corpora luteal function in healthy women. Fertil Steril. 1988;49:620-25. 7. Liu L, Southers JL, Banks SM, et al. Stimulation of testosterone production in the cynomolgus monkey in vivo by deglycosylated and desialylated human choriogonadotropin. Endocrinology. 1989;124:175-80. 8. Auletta FJ, Kamps DL, Pories S, Bisset J, Gibson M. An intracorpus luteum site for the luteolytic action of prostaglandin Fia in
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15. 16.
17. 18.
the rhesus monkey. Prostaglandins. 1984;27:285-98.
9. Sargent EL, Baughman WL, Novy MJ, Stouffer RL. Intraluteal infusion of a prostaglandin synthesis inhibitor, sodium meclofenamate, causes premature luteolysis in rhesus monkeys. Endocrinology. 1988;123:2261-9. 10. Lee CY, Ryan RJ. Interaction of ovarian receptors with human luteinizing hormone and human chorionic gonadotropin. Biochemistry. 1973;12:4609-15. 11. Molskness TA, VandeVoort CA, Stouffer RL. Stimulatory and inhibitory effects of prostaglandins on the gonadotropin-sensitive adenylate cyclase in the monkey corpus luteum. Prostaglandins. 1987;34:279-90. 12. Resko JA, Ploem JG, Stadelman HL. Estrogens in fetal and maternal plasma of the rhesus monkey. Endocrinology. 1975;97:42530. 13. Resko JA, Norman RL, Niswender GD, Spies HG. The relationship
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20. 21.
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between progestins and gonadotropins during the late luteal phase of the menstrual cycle in rhesus monkeys. Endocrinology. 1974;94:128-35. Weick RF, Dierschke DJ, Karsch FJ, Butler WR, Hotchkiss J, Knobil E. Periovulatory time courses of circulating gonadotropic and ovarian hormones in the rhesus monkey. Endocrinology. 1973;93:1140-7. Bourne GH. Collected anatomical and physiological data from the rhesus monkey. In: Bourne GH, ed. The rhesus monkey. New York: Academic Press; 1975;1:1. Groff TR, Madhwa Raj HG, Talbert LM, Willis DL. Effects of neutralization of luteinizing hormone on corpus luteum function and cyclicity in Macaca fascicularis. J Clin Endocrinol Metab. 1984;59:1054-7. Moudgal NR, MacDonald GJ, Greep RO. Effect of hCG antiserum on ovulation and corpus luteum function in the monkey (Macaca fascicularis). J Clin Endocrinol Metab. 1971;32:579-81. Fraser HM, Baird DT, McRae GI, Nestor JJ, Vickery BH. Suppression of luteal function by an LH-RH antagonist during the early luteal phase in the stumptailed macaque monkey and the effects on subsequent administration of hCG. Endocrinology. 1987;121:612-8. Hutchison JS, Zeleznik AS. The rhesus monkey corpus luteum is dependent on pituitary gonadotropin secretion throughout the luteal phase of the menstrual cycle. Endocrinology. 1984;115:17806. Asch RH, Abou-Samra M, Braunstein GD, Pauerstein CJ. Luteal function in hypophysectomized rhesus monkeys. J Clin Endocrinol Metab. 1982;55:154-61. Balmaceda JP, Borghi MR, Coy DH, Schally AV, Asch RH. Suppression of postovulatory gonadotropin levels does not affect corpus luteum function in rhesus monkeys. J Clin Endocrinol Metab. 1983;57:866-8. Chen H-C, Shimohigashi Y, Dufau ML, Catt KJ. Characterization and biological properties of chemically deglycosylated human chorionic gonadotropin. J Biol Chem. 1982;257:14446-52.
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Vol. 70, No. 4 Printed in U.S.A.
The Effect of Intraluteal Infusion of Deglycosylated Human Chorionic Gonadotropin on the Corpus Luteum in Rhesus Monkeys* PHILLIP E. PATTON, RICHARD L. STOUFFER, AND MARY B. ZELINSKI-WOOTEN Department of Obstetrics and Gynecology, Oregon Health Sciences Center (P.E.P.), Portland, Oregon 97201; and the Division of Reproductive Biology and Behavior, Oregon Regional Primate Research Center (R.L.S., M.B.Z.), Beaverton, Oregon 97006
ABSTRACT. Removal of the carbohydrates from hCG results in an antagonist (degly-hCG) that competitively inhibits hCG/ LH-stimulated adenylate cyclase in macaque luteal tissue in vitro, but its effect in vivo is controversial. To examine the effect of degly-hCG on the lifespan and steroidogenic activity of the primate corpus luteum, the antagonist was administered to female rhesus monkeys (n = 7) beginning at the midluteal phase of the menstrual cycle. In a control cycle the saline vehicle was infused via an osmotic minipump directly into the corpus luteum. In a subsequent cycle, one of three dose rates of degly-hCG (0.001, 0.009, and 0.09 nmol/h) was infused into the corpus luteum. Pump implantation and infusion began 5-9 days after the midcycle LH surge and continued for 7 days. Peripheral
H
venous blood was collected daily from day 8 of the cycle until menses, and serum progesterone levels were determined by RIA. Progesterone levels and patterns were similar in animals that received either the saline vehicle or degly-hCG, and the length of the luteal phase in monkeys receiving any dose of degly-hCG (16.4 ± 0.5 days) was not different from that in animals receiving a control infusion (16.1 ± 0.9 days). In a corollary study, an intraluteal infusion of degly-hCG (0.009 nmol/h) in the midluteal phase did not prevent stimulation of progesterone levels after im injection of hCG (15 IU/day for 5 days). We conclude that whereas degly-hCG is a useful tool to examine gonadotropin action in vitro, it is not a potent gonadotropin antagonist in vivo. (J Clin Endocrinol Metab 70: 1213-1218, 1990)
CG AND LH contain a series of complex carbohydrate side-chains attached to amino acid residues in both the a- and /?-subunits. Because removal of the carbohydrate decreases the in vitro action of gonadotropic hormones, the oligosaccharide moieties are considered obligatory for the full expression of biological activity. Chemical deglycosylation of purified hCG results in a stable compound (degly-hCG) with 85% of its carbohydrate residues removed, which acts as an effective antagonist of gonadotropin action in rodent luteal tissue in vitro (1). Moreover, degly-hCG inhibits LH/ CG-stimulated cAMP and steroid production in primate luteal tissue in vitro (2, 3). Despite evidence that degly-hCG acts as an effective gonadotropin antagonist in vitro, few studies have assessed the in vivo effects of degly-hCG on gonadotropindependent tissues. In the rodent, the administration of
degly-hCG apparently interferes with gonadotropindependent ovulation, implantation, and early pregnancy (4, 5). Alternatively, the systemic administration of degly-hCG in the primate has not been shown to inhibit the response to endogenous gonadotropins in LH/CGdependent tissues (6, 7). Nevertheless, the short term infusion (6) and the rapid metabolic clearance of the compound could have limited the antagonism of deglyhCG on gonadotropin tissues. Recently, methods to deliver substances directly to the corpus luteum were developed (8, 9), which circumvents the problems associated with systemic administration. To examine further the in vivo action of degly-hCG on primate luteal function, the antagonist was infused chronically and directly into the corpus luteum of rhesus monkeys beginning at the midluteal phase of the menstrual cycle.
Received August 17,1989. Address requests for reprints to: Phillip E. Patton, M.D., Department of Obstetrics and Gynecology, L-466, Oregon Health Sciences University, 3181 SW Sam Jackson Park Road, Portland, Oregon 97201. * This work was supported by the Medical Research Foundation of Oregon (tp P.E.P.), NIH Grant HD-20869 (to R.L.S.), and NIH Grants HD-18185 and RR-00163. Oregon Regional Primate Research Center Publication 1697.
Materials and Methods degly-hCG
Degly-hCG prepared by chemical treatment of purified hCG was a gift from Dr. Robert Ryan (Mayo Clinic, Rochester, MN). The preparation and in vitro characterization of the compound 1213
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have been described in detail previously (1). No demonstrable agonist activity was apparent when cAMP production was measured in rat ovarian membranes exposed to doses of deglyhCG up to 104 pmol (10). Experimental design The housing and general care of rhesus monkeys at the Oregon Regional Primate Research Center were described previously (11). Monkeys were checked daily for menses, and menstrual records were maintained. Adult females exhibiting normal menstrual cycles of approximately 28 days were used in the study. Saphenous blood samples were drawn daily between 08000830 h from day 8 of the menstrual cycle until menses or pump removal, whichever occurred later. Serum estradiol and progesterone concentrations were measured in the Core Hormone Assay Laboratory at the Oregon Regional Primate Research Center using previously described RIA methodology (12, 13). The intra- and interassay variations were 8.3% and 9.8% for estradiol and 10.7% and 16.5% for progesterone, respectively. An osmotic minipump (Alzet, model 2ML1, Alza Corp., Palo Alto, CA), which delivers between 9.5-10.5 juL/h continuously for 7 days, was implanted sc in the flank of female rhesus monkeys during the midluteal phase (5-9 days after the LH surge) of the menstrual cycle. The day of the LH surge was estimated to occur within the same 24-h interval of the peak preovulatory estrogen level (14). The mean day of pump implantation did not differ significantly among control and treatment groups. The description of the anesthesia and surgical procedures used for pump implantation has been detailed previously (9). The osmotic minipump was secured sc, and a PV4 catheter connected to the pump and ending in a 21-gauge needle was inserted directly into the corpus luteum. Each osmotic minipump was removed 7 days after placement, and the pump reservoirs were inspected to confirm delivery of the infusate. The needle was removed from the ovary during a subsequent laparotomy, generally within 3 months. The study design was such that each animal (n = 7) received an intraluteal infusion of vehicle during a control cycle before a degly-hCG treatment cycle. The saline vehicle consisted of 0.15 M NaCl, pH 7.4, and 0.1% BSA (wt/vol; Sigma Chemical Co., St. Louis, MO). Only animals that exhibited a normal progesterone profile both before and after pump placement in the luteal phase were tested in subsequent treatment cycles. A normal progesterone profile was defined as progesterone values within the 95% confidence interval of progesterone values from comparable days of the menstrual cycle in untreated animals (n = 18) from our colony (10). After the control cycle, one of three dose levels of degly-hCG [0.001 nmol/h (n = 2), 0.009 nmol/h (n = 3), and 0.09 nmol/h (n = 3)] was infused into the corpus luteum during the treatment cycle. In the calculations, the following estimated mol wt were used: 40K for hCG and 31.6K for degly-hCG. The intraluteal dose of degly-hCG necessary to act as a competitive antagonist of LH was estimated from peripheral LH levels and blood flow to the macaque ovary at the midluteal phase of the cycle (15). In a corollary study, either the saline vehicle (n = 1) or degly-hCG (0.009 nmol/h; n = 1) was infused into the
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corpus luteum during the midluteal phase as described previously. Two days after pump implantation, intact hCG (Ayerst APL, Rouses Point, NY; 15 IU/day) was given im for a total of 5 days in each animal. In a final study, intact hCG (15 IU/day) was infused into the corpus luteum of two monkeys as previously described (9). Adenylate cyclose assay Luteinized ovaries of superovulated rats were pooled, homogenized, and centrifuged to obtain a membrane particulate fraction, according to methods previously described (2). The activity of adenylate cyclase was examined using established procedures (2), and activity was assessed in the presence and absence (basal activity) of maximally stimulating doses of GTP (2.5 nmol) and hCG (12.5 pmol; CR123). In addition, freshly prepared degly-hCG (12.5 pmol) and degly-hCG recovered from a minipump after 7 days in vivo (47.5 pmol) were incubated with rat luteal membranes in the absence and presence of 12.5 pmol hCG. The activity evoked by each addition was assayed in triplicate, and the results were expressed as the mean ± SEM. Data were expressed as moles of cAMP produced per min/mg protein. Statistical analysis Daily progesterone levels from animals that received the saline vehicle infusion or intraluteal degly-hCG at the three dose levels tested were analyzed using two-way analysis of variance with repeated measures over time. Luteal phase lengths from the same groups were analyzed by one-way analysis of variance. Differences were considered significant at a level of P< 0.05.
Results Progesterone patterns in monkeys receiving an infusion of the saline vehicle beginning at the midluteal phase of the cycle are shown in Fig. 1. As noted previously (10), serum progesterone levels were lower in the sample obtained 24 h after surgery, but levels in the postpump implantation interval remained in the normal range compared to those in untreated animals. In one of the seven monkeys, progesterone values dropped precipitously, followed by menses on the third day after pump implantation and infusion with the saline vehicle (data not shown). When tested in a subsequent control cycle (data included in Fig. 1), a normal progesterone profile and luteal phase length were noted in this animal. The effect of an intraluteal infusion of degly-hCG at the highest dose tested (0.09 nmol/h) on progesterone profiles is illustrated in Fig. 2. Progesterone profiles obtained with lower infusion rates were similar; therefore, a single representative figure (Fig. 2) is included. As in the saline vehicle-treated monkeys, progesterone levels declined at 24 h, but remained within the normal range during the infusion interval. At all three dose levels tested there were no differences in progesterone levels in
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Days Relative to Pump Implantation FIG. 1. Patterns and levels (mean ± SEM; n = 7) of progesterone in monkeys receiving intraluteal infusions of 0.9% normal saline and 0.1% BSA. Data are normalized to the day of pump implantation (day 0). The horizontal bar on the x-axis indicates the treatment interval. The shaded area represents the 95% confidence interval for serum progesterone levels during this time in the menstrual cycle based on values from untreated animals (n = 18) in our colony. To convert the results to SI units, multiply nanograms per mL P by 3.18 nmol/L.
animals treated with degly-hCG vs. the saline vehicle. As summarized in Table 1, the interval from pump implantation until menses or the length of the luteal phase was not significantly different between animals that received either the saline vehicle or any of the three dose levels of degly-hCG tested. Figure 3 illustrates the progesterone profiles from two monkeys that received either an intraluteal infusion of saline vehicle or degly-hCG (0.009 nmol/h). Both animals received exogenous hCG (15 IU/day, im) for 5 consecutive days, beginning 2 days after pump implantation. Each animal exhibited a similar agonist response to hCG, as reflected by an increase in circulating levels of progesterone within 24 h to 3-fold above the normal range, which was sustained for 4-5 days. The interval from pump implantation until menses was 8 days for each animal. Similarly, the infusion of antagonist for 48 h before the injection of hCG had no effect on the length of the luteal phase compared to that in the saline vehicle-
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Days Relative to Pump Implantation FIG. 2. Patterns and levels (mean ± SEM) of progesterone in monkeys receiving an intraluteal infusion of degly-hCG (0.09 nmol/h; n = 3). Refer to Fig. 1 for details. TABLE 1. Interval between pump placement and menses and length of the luteal phase Intraluteal infusate Saline vehicle (n = 7) Degly-hCG 0.001 nmol/h (n = 2) 0.009 nmol/h (n = 3) 0.09 nmol/h (n = 3)
Interval to menses
Luteal phase length0
9.7 ± 1.76
16.1 ± 0.9*
11.0 8.0 ± 1.0 10.3 ± 2.1
17.0 15.3 ± 0.6 17.0 ± 2.0
Values are the mean (days) ± SEM. ° Day of LH surge to onset of menses. b There were no differences in the interval from pump placement to mense or length of the luteal phase among treatment groups.
treated animal (15 and 16 days, respectively). The effect of an intraluteal infusion of intact hCG on progesterone profiles is depicted in Fig. 4. In contrast to infusions of saline vehicle or degly-hCG (Figs. 1 and 2), there was no apparent drop in progesterone levels 24 h after pump implantation. Using a dose of hCG (15 IU/ day) comparable to the im dose in the previous study,
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Days Relative to Pump Implantation Days Relative to Pump Implantation FIG. 3. Progesterone levels in monkeys receiving an intraluteal infusion of saline (O-O) or degly-hCG (•- -•; 0.009 nmol/h). Both animals received hCG (15 IU/day) for 5 consecutive days, beginning 2 days after pump placement (hCG, 15 IU/day in box).
progesterone levels increased within 24 h of infusion and remained elevated above the normal range for 4-5 days before declining. Table 2 summarizes adenylate cyclase activity in rat luteal membranes in the absence and presence of intact hCG and degly-hCG recovered 7 days after pump implantation in vivo. Both freshly prepared degly-hCG and degly-hCG recovered from the minipump failed to stimulate cAMP production and inhibited hCG-stimulated cAMP production by rat luteal homogenates.
Discussion The primate corpus luteum in the nonfertile cycle appears to be dependent upon pulsatile exposure to pituitary LH. Investigators report an early demise of the corpus luteum when anti-LH antiserum, anti-CG antiserum, or GnRH antagonists are administered to women or monkeys during the luteal phase (16-18). Similarly, using the hypothalamic clamped monkey model with bilateral lesions in the arcuate region of the hypothala-
FIG. 4. Progesterone levels in two monkeys (D and • ) receiving intraluteal infusions of hCG (15 IU/day). Refer to Fig. 1 for details. TABLE 2. Adenylate cyclase activity of rat luteal membranes in the absence and presence of intact hCG, degly-hCG, and degly-hCG recovered 7 days after pump implantation in vivo
Degly-hCG 0 12.5 pmol (fresh) 47.5 pmol (postpump)
cAMP production (pmol/min protein) Control0
12.5 pmol hCG
10.2 ± 0.3 11.6 ± 0.8 9.0 ± 0.5
31.6 ± 2.2 20.4 ± 1.66 16.2 ± 2.2*
Values are the mean ± SEM of triplicate determinations. GTP (2.5 nmol) was present in control and hCG treatment groups (basal activity, 5.8 ± 0.7). b Both freshly prepared degly-hCG and degly-hCG recovered from an osmotic minipump after 7 days in vivo inhibited hCG-stimulated cAMP production by rat luteal homogenates. 0
mus, the withdrawal of GnRH-driven gonadotropin support in the midluteal phase of the menstrual cycle results in a prompt reduction of progesterone and menses (19). Although these studies support a gonadotropin-dependent corpus luteum, there is concern that the substances used or the model employed may include an effect other than an anti-LH action, which influences the function
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or lifespan of the corpus luteum. Furthermore, in view of studies that propose a gonadotropin-independent corpus luteum (20, 21), the mechanisms regulating corpus luteum function remain unresolved. We employed a more direct method of testing the dependence of the corpus luteum on gonadotropins in vivo by locally infusing a substance (degly-hCG) that has been shown to be a gonadotropin antagonist in primate tissues in vitro (2, 3). We hypothesized that the administration of degly-hCG would occupy the luteal cell LH receptor, prevent binding of circulating LH, diminish progesterone secretion, and induce early menses. Nevertheless, infusion of the LH/hCG antagonist into the corpus luteum beginning in the midluteal phase of the menstrual cycle resulted in no change in progesterone levels or luteal phase length compared to those in a control group that received an infusion of saline vehicle. At all dose levels tested there was no evidence that deglyhCG acted as an effective gonadotropin antagonist in macaque luteal tissue in vivo. The failure of degly-hCG to induce early menses is not likely to be due to a subtherapeutic dose, inadequate delivery or distribution of the compound to the corpus luteum, or instability of the compound. First, peripheral serum LH levels (mean ± SEM) measured by bioassay in the rhesus monkey approximate 8.3 ±3.5 ng/mL (using NIH RP-1 standard) in the luteal phase of the menstrual cycle (10). Assuming an ovarian blood supply of approximately 100 mL/h in the luteal phase (16), a 0.09 nmol/ h dose of degly-hCG should deliver approximately a 3fold molar excess of degly-hCG compared to LH to the luteal cell. At the highest concentration used (0.09 nmol/ h), 2.16 nmol degly-hCG would be directly delivered to the corpus luteum per day. Second, it is also unlikely that degly-hCG failed to act as an antagonist because of impaired delivery or poor distribution to the corpus luteum. In two monkeys, intraluteal infusion of a low dose of hCG resulted in a prompt increase in progesterone levels above values in control cycles. Moreover, the generation of antibodies to CG-like molecules by two of three animals (data not shown) implies that the compound was infused in significant quantities. Furthermore, it is doubtful that our results can be explained by instability of the degly-hCG over the interval of infusion. Degly-hCG recovered from the minipump after 7 days in vivo inhibited hCG-stimulated adenylate cyclase activity in rat ovarian membrane homogenates in vitro. The results of our preliminary studies indicated that degly-hCG was not a potent gonadotropin antagonist in vivo. However, the possibility that the corpus luteum is independent of LH support could not be excluded. Therefore, we examined the response of the corpus luteum to exogenous hCG after preexposure to degly-hCG. Previous studies indicated that the incubation of ovarian
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membranes with increasing concentrations of degly-hCG progressively diminishes the adenylate cyclase response to hCG in vitro (2). Nevertheless, the infusion of deglyhCG at a dose of 0.009 nmol/h for 48 h did not inhibit the agonist action of low dose hCG (15 IU/day, im) on corpus luteum function in vivo. Progesterone profiles and luteal phase lengths after exogenous gonadotropin treatment were indistinguishable in monkeys pretreated with degly-hCG or saline vehicle. In contrast to in vitro studies, our results indicate that the infusion of degly-hCG has no effect on basal (LH-supported) or hCG-stimulated corpus luteum function during the menstrual cycle. We conclude that whereas degly-hCG is a useful tool to examine gonadotropin action in vitro, it is not a potent gonadotropin antagonist in primate luteal tissue in vivo. Additional observations support the finding that deglyhCG is not an effective LH/hCG antagonist in primate species in vivo. In women, the peripheral infusion of doses as high as 0.25 nmol/kg-day degly-hCG during the midluteal phase of the menstrual cycle had no effect on daily progesterone levels or the duration of the luteal phase (6). Moreover, a single iv injection of degly-hCG (0.32 ± 0.02 nmol/kg) into the male macaque actually led to an increase in testosterone levels compared to the response to an equimolar dose of hCG, suggesting an agonist effect on Ley dig cell steroidogenesis (7). Therefore, it is possible in the current study that residual agonistic activity of degly-hCG maintains the corpus luteum despite antagonism of circulating gonadotropins, as observed in the male macaque in vivo and gonadotropin-dependent tissues in vitro (3, 22). In summary, the infusion of degly-hCG directly into the corpus luteum had no effect on the patterns of circulating progesterone or the length of the luteal phase in rhesus monkeys. Thusfar, the data obtained after bolus injection, short term peripheral infusion, or chronic intraluteal infusion provide no substantial evidence of antagonism of gonadotropin-stimulated action by deglyhCG in primates in vivo. It is doubtful that degly-hCG will prove to be an effective luteolytic agent in primates in the future. Acknowledgments We would like to thank Dr. Robert Ryan for the generous gift of degly-hCG, and Dr. David Hess and his staff for performing steroid assays. Special thanks are due to Dr. Ted Molskness for technical advice and support, and Elizabeth Cook for her secretarial services.
References 1. Keutmann HT, Mcllroy PJ, Bergert ER, Ryan RJ. Chemically deglycosylated human chorionic gonadotropin subunits: characterization and biological properties. Biochemistry. 1983;22:3067-72. 2. Eyster KM, Stouffer RL. Adenylate cyclase in the corpus luteum of the rhesus monkey. Sensitivity to nucleotide, gonadotropins, catecholamines, and nonhormanal activators Endocrinology.
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1985;116:1552-8. 3. Richardson MC, Masson GM, Sairam MR. Inhibitory action of chemically deglycosylated human chorionic gonadotropin on hormone-induced steroid production by dispersed cells from human corpus luteum. J Endocrinol. 1984;101:327-32. 4. Kato K, Sairam MR, Manjunath P. Inhibition of implantation and termination of pregnancy in the rat by a human chorionic gonadotropin antagonist. Endocrinology. 1983; 113:195-99. 5. Kato K, Sairam MR. Inhibition of ovulation in the rat by an hCG antagonist. Contraception. 1983;27:515-20. 6. Patton PE, Calvo FO, Fujimoto VY, Bergert ER, Kempers RD, Ryan RJ. The effect of deglycosylated human chorionic gonadotropin on corpora luteal function in healthy women. Fertil Steril. 1988;49:620-25. 7. Liu L, Southers JL, Banks SM, et al. Stimulation of testosterone production in the cynomolgus monkey in vivo by deglycosylated and desialylated human choriogonadotropin. Endocrinology. 1989;124:175-80. 8. Auletta FJ, Kamps DL, Pories S, Bisset J, Gibson M. An intracorpus luteum site for the luteolytic action of prostaglandin Fia in
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the rhesus monkey. Prostaglandins. 1984;27:285-98.
9. Sargent EL, Baughman WL, Novy MJ, Stouffer RL. Intraluteal infusion of a prostaglandin synthesis inhibitor, sodium meclofenamate, causes premature luteolysis in rhesus monkeys. Endocrinology. 1988;123:2261-9. 10. Lee CY, Ryan RJ. Interaction of ovarian receptors with human luteinizing hormone and human chorionic gonadotropin. Biochemistry. 1973;12:4609-15. 11. Molskness TA, VandeVoort CA, Stouffer RL. Stimulatory and inhibitory effects of prostaglandins on the gonadotropin-sensitive adenylate cyclase in the monkey corpus luteum. Prostaglandins. 1987;34:279-90. 12. Resko JA, Ploem JG, Stadelman HL. Estrogens in fetal and maternal plasma of the rhesus monkey. Endocrinology. 1975;97:42530. 13. Resko JA, Norman RL, Niswender GD, Spies HG. The relationship
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between progestins and gonadotropins during the late luteal phase of the menstrual cycle in rhesus monkeys. Endocrinology. 1974;94:128-35. Weick RF, Dierschke DJ, Karsch FJ, Butler WR, Hotchkiss J, Knobil E. Periovulatory time courses of circulating gonadotropic and ovarian hormones in the rhesus monkey. Endocrinology. 1973;93:1140-7. Bourne GH. Collected anatomical and physiological data from the rhesus monkey. In: Bourne GH, ed. The rhesus monkey. New York: Academic Press; 1975;1:1. Groff TR, Madhwa Raj HG, Talbert LM, Willis DL. Effects of neutralization of luteinizing hormone on corpus luteum function and cyclicity in Macaca fascicularis. J Clin Endocrinol Metab. 1984;59:1054-7. Moudgal NR, MacDonald GJ, Greep RO. Effect of hCG antiserum on ovulation and corpus luteum function in the monkey (Macaca fascicularis). J Clin Endocrinol Metab. 1971;32:579-81. Fraser HM, Baird DT, McRae GI, Nestor JJ, Vickery BH. Suppression of luteal function by an LH-RH antagonist during the early luteal phase in the stumptailed macaque monkey and the effects on subsequent administration of hCG. Endocrinology. 1987;121:612-8. Hutchison JS, Zeleznik AS. The rhesus monkey corpus luteum is dependent on pituitary gonadotropin secretion throughout the luteal phase of the menstrual cycle. Endocrinology. 1984;115:17806. Asch RH, Abou-Samra M, Braunstein GD, Pauerstein CJ. Luteal function in hypophysectomized rhesus monkeys. J Clin Endocrinol Metab. 1982;55:154-61. Balmaceda JP, Borghi MR, Coy DH, Schally AV, Asch RH. Suppression of postovulatory gonadotropin levels does not affect corpus luteum function in rhesus monkeys. J Clin Endocrinol Metab. 1983;57:866-8. Chen H-C, Shimohigashi Y, Dufau ML, Catt KJ. Characterization and biological properties of chemically deglycosylated human chorionic gonadotropin. J Biol Chem. 1982;257:14446-52.
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