Active immunization against LHRH: I. Effects of conjugation site and dose.

UNLABELLED: Active immunization against LHRH is a promising method of contraception for men. In order to be acceptable, sufficient amounts of anti-LHRH antibodies must be induced rapidly after vaccination. In previously reported animal studies, we found that it took considerable time (up to 5 months) to obtain antibody titers (AT) that were sufficiently high for complete suppression of spermatogenesis. The possibility of accelerating the immune response to LHRH by increasing the dose of immunogen was investigated in the male rat. Six doses of LHRH conjugated to tetanus toxoid (TT) in the 10 position (LHRH10-TT), ranging from 2.5 to 612 micrograms, and three doses of LHRH1-TT (50 to 612 micrograms) were tested. The magnitude of the immune response did not depend on the dose of the antigen, provided a threshold dose had been surpassed. Antigenicity of LHRH conjugated to TT at either the 1-, 6-, or 10-position was compared in rats and rabbits. In both species LHRH1-TT induced sufficient antibody concentrations to suppress pituitary gonadotropins (LH and FSH) and, subsequently, serum testosterone (T) levels faster than either the 6- or 10-conjugates. Only materials permitted for use in humans were utilized in these experiments. CONCLUSION: Active immunization against LHRH conjugated to TT at the 1-position has potential as a fast, convenient method of male contraception.

The implication of opiates in the glucocorticoid-mediated inhibition of LH secretion in rats.

The present study was designed to test the effect of acute administration of dexamethasone on the postcastration and gonadotrophin-releasing hormone (GnRH)-induced rise of LH, and to examine whether the inhibitory action of glucocorticoids on LH secretion was mediated by the opioid system. Rats were castrated and injected 10-10.5 h later in a first set of experiments with saline, dexamethasone (250 micrograms/rat), nalmefene (2 mg/kg body weight) or nalmefene plus dexamethasone. The response to GnRH was tested 11 h after castration (time 0) in all groups. Dexamethasone caused a significant (P less than 0.005) decrease in basal serum concentrations of LH, while saline and nalmefene did not induce any change. The administration of dexamethasone preceded by nalmefene increased basal concentrations of LH (1.6 times), with an effect significantly greater than that of dexamethasone (P less than 0.001), nalmefene (P less than 0.05) or saline (P less than 0.005) alone. GnRH induced a significant (P less than 0.001) increase in serum concentrations of LH in all groups. In a second set of experiments, administration of naloxone (2 mg/kg body weight) increased LH levels (P less than 0.05) and similarly reversed the inhibitory effect of dexamethasone on LH.(ABSTRACT TRUNCATED AT 250 WORDS)

Urinary steroid concentrations during natural and gonadotrophin-induced oestrus and pregnancy in the giant panda (Ailuropoda melanoleuca).

Urinary concentrations of conjugated oestrone and pregnanediol-3-glucuronide were measured during and after spontaneous and induced oestrus and during pregnancy. Behavioural oestrus was preceded by a rise in oestrone values from less than 10 ng/mg creatinine (Cr) to peaks of 45 ng/mg Cr. Maximal lordotic response and mating activity coincided with the decline in oestrone levels. After presumed ovulation, urinary pregnanediol glucuronide concentrations increased from less than 5 to 15-30 ng/mg Cr. Further increases in this steroid (to 60-80 ng/mg Cr) occurred 114 days after mating, presumably coincident with implantation. These high levels of pregnanediol glucuronide were maintained for 3 weeks, began to decline 1 week before parturition and fell to a nadir (less than 5 ng/mg Cr) immediately after delivery. When FSH was administered i.m. for 5 days, urinary oestrone values rose markedly and were maximal (580 ng/mg Cr) on Day 7. Mating first occurred on Day 20 and 500 i.u. hCG were given i.m. Urinary pregnanediol glucuronide levels during the next 5 months were similar to those in the previous year during pregnancy with values rising 105-108 days after mating. However, no birth occurred. These results support the suggestion that pandas exhibit delayed implantation and demonstrate that the panda is responsive to exogenous gonadotrophins.

Active immunization against gonadotropin-releasing hormone combined with androgen supplementation is a promising antifertility vaccine for males.

Male rats and rabbits were immunized against gonadotropin-releasing hormone (GnRH) conjugated to tetanus toxoid (GnRH10-TT) using only materials approved for humans. Testosterone (T)-releasing implants or the long-lasting T ester testosterone-17-trans-4-n-butyl-cyclohexane carboxylate (TE) was used as supplemental androgen for maintaining libido. Immunization against GnRH10-TT effectively suppressed fertility (spermatogenesis) in rats and rabbits. Neither T nor TE administration restored fertility. Both androgens were effective in maintaining normal libido in rats. TE, which is not hydrolyzed in rabbits, was less effective in maintaining normal ejaculatory behavior in this species. Active immunization against GnRH could be a convenient and cost-effective method of fertility control in males.

Failure of gonadotropin therapy secondary to chorionic gonadotropin-induced antibodies.

Seventeen years after first receiving treatment with hCG (at age 8 yr), a man with hypogonadotropic hypogonadism no longer responded to gonadotropin therapy. He had received hCG for 6 months when he was 8 yr old, from age 18-21 yr and from age 21-25 yr, when the resistance developed. Anti-hCG antibodies were found in his serum. Three sequential treatment regimens were tried to obviate the effect of these antibodies. 1) hCG treatment (2000 IU, three times per week) concomitant with weekly plasmapheresis (since the patient's response to an hCG challenge test was improved after a reduction of antibody titer by plasmapheresis) resulted in only a temporary increase in testosterone production. 2) Treatment with human (h) LH (400 IU/week) and hFSH (25 IU/week) was used because of the low cross-reaction of the antibodies with hLH and a response to a hLH-challenge test. This treatment maintained serum testosterone levels within the normal range for long periods, but had to be discontinued when the supply of hLH was exhausted. 3) Pulsatile LHRH administration (25 ng/kg, sc, every 2 h) for 2 months did not induce the release of pituitary gonadotropins. These results indicated that 1) conventional hCG treatment was impaired by antibody-induced changes in the kinetics of hCG after its im administration; 2) hLH was an effective substitute for hCG, and the combined hLH-hFSH administration initiated a moderate amount of spermatogenesis; and 3) the patient differs from most individuals with hypogonadotropin hypogonadism in that he did not have normal responses to repetitive LHRH administration.

Long-term immunization against the beta-subunit of ovine luteinizing hormone (oLH beta) has no adverse effects on pituitary function in rhesus monkeys.

One postulated safety hazard of contraceptive methods based on immunization against gonadotropic hormones is the possibility that circulating antibodies which crossreact with pituitary hormones may impair pituitary function through the deposition of immunoglobulin and/or complement suggesting immune complexes. In order to evaluate this possibility in rhesus monkeys actively immunized against the beta-subunit of ovine luteinizing hormone (oLH beta), we used three approaches to study the effects of long-term immunization on pituitary function: a) evaluation of pituitary responsiveness to challenge with a GnRH-agonist; b) examination of pituitary histology and immunostaining with gonadotropin antisera; and c) examination of pituitary cells for deposition of immune complexes. Our results indicate that circulating anti-oLH beta antibodies did not result in significant impairment of pituitary function in rhesus monkeys.

Evidence suggesting that germ cells influence the bidirectional secretion of androgen binding protein by the seminiferous epithelium demonstrated by selective impairment of spermatogenesis with busulphan.

Androgen binding protein (ABP) was measured in the serum, testes and epididymides of adult rats up to 105 days after the induction of reversible impairment of spermatogenesis by a single injection of busulphan. This treatment decreased testicular and epididymal weights within 7-21 days after treatment, reaching a minimum at 63 days with partial recovery by 105 days. The testicular and epididymal content of sperm was unchanged up to 42 days after busulphan administration, was reduced considerably at 63 days and thereafter increased towards control values. The serum and testicular concentrations of testosterone were normal at all times after treatment, even though serum LH levels were increased at 42 and 63 days. Serum levels of FSH were also increased at 43 and 63 days after treatment. A biphasic pattern in the serum levels of ABP was observed. Concentrations were low up to 43 days post treatment when only the early germ cell types were depleted from the seminiferous epithelium and when the testicular and epididymal contents of ABP were normal. Serum levels of ABP increased as the more mature germ cells were depleted in numbers and the testicular and epididymal contents of ABP declined. It is concluded that bidirectional secretion of ABP into the interstitium (serum) and into the seminiferous tubular lumen by Sertoli cells is influenced considerably by the population of germ cells that are present in the seminiferous epithelium.

Experimental evidence supporting a mathematical theory of the physiological mechanism regulating follicle development and ovulation number.

In higher vertebrates, follicular development is regulated so that the number of follicles that periodically mature and ovulate is controlled within a narrow range. Lacker has proposed a simple mathematical model of follicle development that can account for the regulation of ovulation number. To support the assumption of the theory that follicle interactions are mediated by estradiol acting as a chemical messenger to communicate follicular maturity to the pituitary and other follicles, we have presented data to demonstrate that in the rabbit physiological concentrations of circulating estradiol inhibit follicle maturation. Implants containing estradiol were placed subcutaneously after surgical rupture of the existing follicles that were 1 mm in diameter or larger. Serum estradiol concentrations were maintained near physiological concentrations by the implants. Concentrations of circulating estradiol were 74 +/- 5.7 pg/ml in the untreated groups, whereas the concentrations with the implants were increased by approximately 50 pg/ml/implant over this basal concentration with a range of 100-300 pg/ml. In the control groups, the average number of follicles before surgical rupture was 27 +/- 2.9 and there was no significant difference (p greater than 0.05) in the number of follicles, 26 +/- 1.9, three days after follicle rupture. The follicles ranged in size from 1 mm to 4 mm, and only those over 3 mm were considered mature. In the first group of animals with implants, the total number of follicles before surgery was 19 +/- 3; three days after follicle rupture, the number of follicles was only 9 +/- 1.1.(ABSTRACT TRUNCATED AT 250 WORDS)

Reversal of testicular function after prolonged suppression with an LHRH agonist in rhesus monkeys.

Using subcutaneously implanted osmotic pumps, four male rhesus monkeys were continuously infused for 18 months with 100 micrograms/day of [(imBzl)-D-His6-Pro9-NEt]-LHRH (LHRH-A), a potent agonist of LHRH. After an initial increase, serum testosterone levels declined to 10% of pretreatment levels in three monkeys and the response to electroejaculation was lost. There was a decrease in testicular volume. Androgen replacement in the form of subcutaneous SILASTIC implants releasing 7 alpha-methyl-19-nor-testosterone acetate led to a restoration of ejaculatory response and the electroejaculates were devoid of spermatozoa. Under this treatment regimen (100 micrograms LHRH-A + 100 micrograms androgen daily), azoospermia was essentially maintained in the three monkeys for about 8 months. Withdrawal of LHRH-A and androgen treatment led to a complete restoration of testicular function. Serum testosterone returned to control levels and spermatozoa reappeared in the ejaculates with sperm counts reaching the normal range. Testicular volumes showed a gradual increase. These results indicate that continuous administration of an LHRH agonist together with an androgen can induce an extended period of azoospermia in rhesus monkeys. These results also show that after prolonged suppression (more than one year) of testicular function complete recovery occurs after cessation of treatment.

Regulation of pituitary gonadotropin-releasing hormone receptors by androgens in the male rabbit.

The regulation of pituitary GnRH receptors was studied in adult male rabbits after castration and androgen replacement with testosterone (T) or 7 alpha-methyl-19-nortestosterone acetate (U-15,614; T analog) supplied by Silastic capsules implanted sc. Castration increased pituitary GnRH receptors significantly, from 99.3 to 329.5 fmol/mg protein within 4 weeks, without a change in the equilibrium association constant. Serum LH concentrations increased from 0.45 to maximum levels of 2.6 ng/ml by day 8 after orchiectomy; these levels persisted throughout the 4 weeks of study. Serum FSH reached maximum levels of 33.6 ng/ml 5 days after castration. T replacement with 250, 500, and 1000 micrograms/kg X day, prevented a postcastration rise in both pituitary GnRH receptor concentrations and gonadotropin secretion, while 100 micrograms/kg X day prevented an increase in GnRH receptors, but did not completely inhibit hypersecretion of gonadotropins. Administration of T analog at doses of 6.25 and 12.5 micrograms/kg X day partially suppressed the castration-induced increase in pituitary GnRH receptor concentrations, while 25, 50, and 100 micrograms/kg X day suppressed GnRH-binding sites to the levels found in intact controls in 15 of 16 rabbits. By contrast, none of the T analog doses was able to prevent completely LH and FSH hypersecretion. The fact that both T and T analog induced dose-dependent stimulation of prostate and seminal vesicle weights indicates that there are tissue-specific differences in the sensitivity to androgens. We conclude that in the male rabbit 1) pituitary GnRH receptors significantly increase after castration; 2) this increase may partially mediate the postcastration hypersecretion of LH and FSH; 3) castration-induced effects can be prevented by androgen replacement. These results are similar to those obtained in rats, where castration increases LHRH receptors, but contrast with results in mice and hamsters, where castration either reduces or does not change receptor levels. This indicates significant species differences in the response of pituitary GnRH receptor concentrations to elimination of the negative feedback effects of androgens.

Unexpected effects of nalmefene, a new opiate antagonist, on the hypothalamic-pituitary-gonadal axis in the male rat.

In order to gain additional information on the role of brain opioid peptides in the regulation of the hypothalamic-pituitary-gonadal axis, we studied the effects of nalmefene, a new opiate antagonist, on gonadotropin and testosterone secretion in male rats. The results were compared with those obtained with naloxone, a well-studied antagonist. Acute injections of either nalmefene or naloxone (2 mg/kg) produced 4-fold increases in LH and testosterone secretion. In castrated male rats treated with testosterone propionate (TP), nalmefene (10 mg/kg) reversed the androgen negative feedback on LH secretion; surprisingly, when higher doses (25 and 50 mg/kg) were injected, the compound lost its ability to antagonize the testosterone-induced inhibition of LH levels. In contrast, naloxone was able to increase LH levels in TP-treated castrated rats even at the highest dose tested (50 mg/kg). Chronic administration of these antagonists resulted in suppression of the acute release of LH and T secretion in nalmefene-treated but not in naloxone-injected animals. These data are consistent with previous observations suggesting that opioid peptides a) exert a tonic inhibitory effect on LH and testosterone production and b) participate in the negative androgen-induced feedback control of LH secretion. Our results also show that the antagonistic action of nalmefene, but not naloxone, is reversed when higher doses are used or following chronic administration.

Testicular cell differentiation in fetal mouse ovaries following transplantation into adult male mice.

Testicular development is a complicated process involving differentiation and arrangement of several cell types. To analyze the process of testicular organization we examined the sequence of the appearance of testicular structures induced in fetal ovaries following transplantation. Fetal mouse ovaries on the twelfth day of gestation were transplanted beneath the kidney capsules of adult male mice. They continued to develop morphologically as ovaries until the eleventh day after transplantation, when seminiferous cord formation and testosterone production began in addition to follicle development (ovotestes). Between the eleventh and fourteenth day after transplantation, ovarian grafts frequently contained transitional structures consisting of Sertoli cells, pregranulosa cells, a third type of cells which show intermediate characteristics between Sertoli and pregranulosa cells, and oocytes enclosed by common basal lamina. Leydig cells or peritubular myoid cells were not found in the transitional area, whereas these cells were present around seminiferous cords composed only of Sertoli cells. Oocytes were absent or degenerated in the well-developed seminiferous cords. The present findings suggest that, in ovarian grafts, pregranulosa cells can differentiate into Sertoli cells, which are responsible for the organization of the seminiferous cords, degeneration of oocytes, and differentiation of other testicular somatic cell types.

Species differences in the sensitivity to GnRH analogs.

The effects of several GnRH agonists and antagonists with high biological activity, have been investigated in rats, mice, rabbits and monkeys. Striking differences exist in the response of different species to the antigonadal and antipituitary effects of these peptides. Of all the animals studied, the rat is the most sensitive. The magnitude of the response to GnRH agonists seems to depend on the sensitivity of the pituitary and the presence of GnRH receptors in the target organs. Findings from animal models require careful interpretation before predictions can be made regarding their possible effects in the human.

Species differences in the sensitivity to a GnRH antagonist.

The effects of the potent GnRH antagonist [Ac-D-NAL(2)1, 4F-D-Phe2, D-Trp3, D-Arg6]-GnRH (GnRH-A), on ovulation in mature rats and rabbits and on serum LH and FSH levels in ovariectomized rats, rabbits and mice were investigated. Dose-response studies showed that 1 microgram (4 micrograms/kg) of GnRH-A was sufficient to inhibit ovulation completely in cycling rats, while 500 micrograms (135 micrograms/kg) were required to inhibit mating induced ovulation in 8 of 11 rabbits. Two of the 3 rabbits which ovulated in spite of the antagonist treatment had delayed LH surges. The mean LH peak of these 3 rabbits was significantly (p less than 0.001) lower than that of controls. Pituitary response to GnRH-A, as measured by plasma gonadotropin levels following GnRH-A treatment in ovariectomized rabbits, rats and mice showed highest sensitivity of the rat to the inhibitory effects of the antagonist. Serum FSH levels were slightly suppressed in rats, but remained undiminished in rabbits and mice. The difference in the response of the three species to the antipituitary effects of GnRH-A is most likely due to differences in the affinity of the pituitary GnRH receptor to the antagonist.

Ovulation inhibition in the pregnant mare's serum gonadotropin-treated immature rat: a bioassay for luteinizing hormone-releasing hormone antagonists.

A convenient method for evaluating the biological activity of luteinizing hormone-releasing hormone (LHRH) antagonists was devised. Pregnant mare's serum gonadotropin (PMSG) treatment of immature rats is known to stimulate follicular growth and estrogen production, that in turn stimulates the release of LHRH which triggers an ovulatory discharge of luteinizing hormone (LH) from the pituitary. The present bioassay of the antagonists is based on the inhibition of ovulation in the PMSG-treated rats. Twenty-eight-day-old Sprague Dawley rats maintained under a light period of 12 h/day (lights on at 0630 h) were given 10 IU of PMSG s.c. at 0930 h. On Day 30 of age the antagonist was given s.c. at 1430 h. The rats were killed on the following morning and the oviducts examined for the presence of ova. In addition, the antagonists were compared in their ability to inhibit serum testosterone levels in adult male rats. In the PMSG-treated rats the order of ovulation-inhibiting potency of the following antagonists was: [Ac-D-NAL(2)1,4FD-Phe2,D-Trp3,D-Arg6]-LHRH (LHRH-1) greater than [Ac-delta 3 Pro1,4FD-Phe2,D-NAL(2)3.6]-LHRH (LHRH-2) greater than [Ac-delta 3 Pro1,4FD-Phe2,D-Trp3,6]-LHRH (LHRH-3). The order of potency was confirmed by their antitesticular effects in adult male rats.

Effect of an LHRH agonist on pituitary and testicular function in rhesus monkeys.

Male rhesus monkeys were given 100 micrograms [(imBzl)-D-His6,Pro9-NEt]-LHRH (LHRH-A), a potent LHRH agonist, s.c. daily for 40 weeks. The first dose of LHRH-A caused acute increases (2-4 h after injection) in serum LH (50-fold), FSH (2 X 5-fold) and testosterone (15-fold) concentrations. Chronic treatment led to a 95% decrease in LH and FSH responses. In spite of a marked decrease in LH response the effect on testosterone response was less evident. Administration of 50 i.u. hCG to control and LHRH-A-treated animals showed that the testicular steroidogenic response was unimpaired by the chronic treatment. Evaluation of the electroejaculated semen at regular intervals showed that there was no consistent reduction in the sperm count of LHRH-A-treated monkeys. Testicular biopsies showed that normal spermatogenesis was occurring in all treated animals, but testicular volume was significantly decreased. These results suggest that, in rhesus monkeys, the pituitary is more susceptible to desensitization by chronic LHRH agonist treatment than are the testes, and that LHRH agonists do not have direct antitesticular effect in rhesus monkeys.

Antibodies against the beta-subunit of ovine luteinizing hormone can decrease the clearance of human chorionic gonadotropin in rhesus monkeys.

Contraceptive vaccines based on active immunization against gonadotropic hormones are being investigated in humans and other primates. Immunization against the beta-subunit of ovine luteinizing hormone (oLH beta) reduces fertility in rhesus monkeys by inducing inadequate luteal phases and preventing corpus luteum rescue by rhesus chorionic gonadotropin (rhCG). These effects result from the cross-reactions of the oLH beta-antibodies with rhCG and rhLH. We used human CG (hCG), which also cross-reacts strongly with anti-oLH beta to examine how the circulating oLH beta-antibodies affect the metabolic clearance rates (MCR) of hCG in rhesus monkeys. 125I-hCG was injected into four nonimmunized and seven immunized monkeys and blood was collected at frequent intervals over 7 days. Total and immunoprecipitable radioactivity did not differ significantly, suggesting that the radioactivity in the plasma consisted almost entirely of 125I-hCG. This was confirmed by column chromatography. The MCR (mean +/- SE) was significantly lower (p less than 0.001) in six immunized monkeys (0.35 +/- 0.06 liters/day) as compared to controls (1.19 +/- 0.09 liters/day). The hCG disappearance curve in control monkeys was best described by a two-compartmental system (slow and fast) while an additional third (intermediate) compartment of distribution was typical for immunized animals. The half-lives of hCG for the two exponentials corresponding to the slow and fast components of distribution were not significantly different between the two groups. One immunized monkey had a MCR (1.44 liters/day) that was much greater than the MCR of the other six.(ABSTRACT TRUNCATED AT 250 WORDS)

[Ac-D-NAL(2)1,4FD-Phe2,D-Trp3,D-Arg6]-LHRH, a potent antagonist of LHRH, produces transient edema and behavioral changes in rats.

Acute toxicity studies of [Ac-D-NAL(2)1,4FD-Phe2,D-Trp3,D-Arg6]-LHRH (LHRH-A), a potent antagonist of LHRH were performed. Subcutaneous administration of this peptide to rats induced transient edema of the face and extremities. This effect was maximal 3-5 h after peptide administration and subsided by 24 h. These effects were not seen with an LHRH agonist or two other antagonists. This side effects of LHRH-A was peculiar to rats and not observed in mice, rabbits and rhesus monkeys. Intravenous administration led within minutes to depression of spontaneous activity in rats and monkeys. We conclude that some LHRH antagonists produce species specific effects on vascular permeability and spontaneous activity.

Species differences in the sensitivity to the antitesticular effects of [Ac-D-NAL(2)1, 4FD-Phe2, D-Trp3,D-Arg6]-LHRH, a potent LHRH antagonist.

The antigonadal effects of [Ac-D-NAL(2)1, 4FD-Phe2, D-TRP3, D-Arg6]-LHRH (LHRH-A), a potent antagonist of LHRH, were investigated in rats and rabbits. Rats and rabbits were given LHRH-A (1250 micrograms/kg) daily for 15 days. Some animals were killed on day 16 (24 h after the last treatment) while others were mated. In the male rats serum LH and testosterone levels as well as the weights of sexual organs were significantly reduced. Mating behavior and fertility that were suppressed by the end of treatment returned to normal by 7 weeks after last treatment. In contrast to rats, the testicular function and fertility of rabbits appeared unaffected by LHRH-A treatment. The difference in the response between rats and rabbits led us to compare the response of rats and mice. Male rats and mice were given LHRH-A (1450 micrograms/kg) daily for 5 days and killed on day 6. In rats LHRH-A caused a 93% decrease in serum T and 88% decrease in in vitro testicular T production. In mice, however, the Leydig cell function remained unaffected when examined 24 h after the last dose of LHRH-A. To explain the differences between the effects of LHRH-A on rats, rabbits and mice, the acute effect of this peptide on serum T levels was investigated in these species. Administration of a single dose of LHRH-A (1250 micrograms/kg) led to a rapid decrease in serum T that was sustained for 24 h in rats. In rabbits and mice, however, the same dose of LHRH-A caused only a transient decrease in serum T. Male rhesus monkeys treated with LHRH-A (1000 micrograms/kg) also showed a transient decrease in serum T concentrations. It is concluded that there are considerable species differences in the sensitivity to the antigonadal effects of LHRH-A.