The effect of active and passive immunization with luteinizing hormone-releasing hormone (LHRH) on serum LH and follicle-stimulating hormone (FSH) levels and the ultrastructure of the pituitary gonadotrophs was investigated in castrated male rats. Two weeks after castration, the animals were immunized with Glu1-LHRH conjugated with human serum albumin (hSA), immunized with hSA only, or left uninjected. Immunogens were administered every 2 weeks. Four weeks after the initiation of immunization with hSA-Glu1-LHRH, 2 out of 4 rats showed parallel decreases in serum LH and FSH levels associated with a rise of serum antibody titer to LHRH. Serum LH and FSH levels remained suppressed throughout the experiment in these rats. On the other hand, both LH and FSH levels in hSA-immunized rats or non-immunized rats remained elevated, and typical castration cells containing large vacuoles were found in the pituitary. Although castration cells existed in the pituitary of rats which produced antibody to LHRH by active immunization, these cells were markedly degranulated, and secretory granules were scarce in the cytoplasm. In another experiment, rats were injected iv with one ml sheep anti-LHRH gamma-globulin (anti-LHRH) or normal sheep gamma-globulin (NSG) every 2 days for 3 weeks, starting one day after castration, when serum LH and FSH levels were already elevated. All the animals which received anti-LHRH showed a decrease in both serum LH and FSH levels, which remained low throughout the study, in a range comparable to those in intact normal male rats. On the other hand, in the animals which received NSG, both LH and FSH levels remained high or increased further throughout the experiment, and the pituitary contained numerous castration cells. Castration cells were completely absent from the pituitaries of rats treated with anti-LHRH, suggesting that castration cells are formed as a result of increased secretion of LHRH. Some FSH gonadotrophs in these castrated rats were atrophic. It was difficult to distinguish the LH gonadotrophs in rats which were either actively or passively immunized with LHRH; however, they seem not to have contributed significantly to the development of castration cells. In any case, antibody to the LHRH decapeptide drastically affected both LH and FSH cells, providing additional evidence for the concept that LHRH represents the physiological LHRH and FSHRH.

The effect of the administration of sheep anti-LHRH gamma-globulin (anti-LHRHG) on the maintenance of pregnancy was investigated in rats. Nidation was confirmed by laparotomy on day 7 or 8 of pregnancy. In one experiment, rats were then injected iv with 1 ml anti-LHRHG or normal sheep gamma-globulin (NSG) daily from days 7 to 11. The uteri were inspected again on day 14 of pregnancy, when it was found that complete resorption of the fetuses had occurred in the anti-LHRHG-treated rats, but that the fetuses were normal in the NSG-treated control rats. The effect of a single injection of 1 ml of anti-LHRHG on day 7, 8, 9, 10, 11, or 12 of pregnancy was also investigated. Administration on day 9 or 10 resulted in complete resorption of the fetuses by the time of the 2nd inspection on day 14, and treatment on day 8 or 11 was partially effective. However, treatment on day 7 or 12 exerted little effect on viability of the fetuses. None of the rats showed vaginal bleeding following treatment with anti-LHRHG. Termination of pregnancy by anti-LHRHG could be prevented by SC injection of 1 mug LHRH twice daily, or by 4 progesterone SC once daily, from days 9 through 12 of pregnancy. The ovaries of the rats treated with anti-LHRHG from days 7 to 11 were smaller than those of the NSG-treated control rats, and some of the corpora lutea underwent cystic degenerative changes. Lutein cells of the former were also smaller than those of the latter. Serum progesterone levels were reduced after a single injection of anti-LHRHG on day 9 or 10, but not on day 7 or 12 of pregnancy. There was excellent agreement between the reduction of serum progesterone and fetal resorption. Serum LH levels were low on days 7 through 12 in the anti-LHRHG-treated as well as the NSG-treated rats, and the possible suppressive effect of anti-LHRHG on LH could not be revealed because of insufficient sensitivity of the radioimmunoassay method. No significant difference was observed in serum prolactin levels between the groups of rats. The results clearly indicate that LHRH, by maintaining progesterone secretion, is indispensable on days 9 and 10 of pregnancy for the maintenance of pregnancy.

One-half ml of sheep-anti-LHRH-serum (No. 772) completely blocked ovulation when administered iv or sc at any stage of the estrous cycle in cycling hamsters. This anti-ovulatory activity lasted 12-13 days. The minimal effective iv dose (MED) of the anti-LHRH serum for completely blocking the preovulatory LH surge and ovulation when administered at 1200 h on proestrus was 0.2 ml. Injection of LHRH on the afternoon of P (proestrus) induced ovulation in the antiserum-treated hamsters when the antiserum was injected on P, but not when injected on D1 (diestrus 1) or D2 (diestrus 2). This suggests that the anti-LHRH serum acts differently in blocking ovulation during D and P, by suppressing follicular development and inhibiting preovulatory surge of gonadotropins, respectively. Serum estradiol levels, measured by radioimmunoassay, were significantly reduced but not completely suppressed after injection of 0.5 ml of anti-LHRH-serum. Histological examinations of the ovaries revealed that an arrest of follicular maturation resulted 3 days after antiserum injection. 17-beta-Estradiol administered 22 h before the presumptive preovulatory LH surge improved significantly (P less than 0.05) the LH response to LHRH in the antiserum-blocked hamsters. This suggests a direct modulation of pituitary LH responsiveness by estradiol in the absence of endogenous LHRH activity. When the integrated levels of serum LH following an injection of a minimum effective dose of exogenous LHRH to induce full ovulation in anti-serum-blocked hamsters were compared with those during the physiological preovulatory LH surge, it was found that only 11% of the amount of LH released on the afternoon of proestrus was sufficient for inducing full ovulation.

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The search continues for inhibitory analogs of LH-RH which have a high affinity for pituitary receptors and better resistance to degradation by plasma and tissue peptidases. Des-His2-desGlyl0-LH-RH ethylamide was the first analog reported to be a potent inhibitor of LH release induced by LH-RH both in vivo with immature male rats (1) or ovariectomized steroid-blocked rats (2) and in vitro (3). Subsequently, other analogs of LH-RH having an enhanced ability to inhibit the LH and FSH released by LH-RH have been reported (4, 5). Recently, D-Phe2-D-Leu6-LH-RH was synthesized in our laboratories based on the inhibitory analog D-Phe2-LH-RH (6) and the powerful gonadotropin-releasing analog D-Leu6-LH-RH (7). It showed a greatly prolonged suppression of LH release in response to LH-RH in immature male rats (8). The surge of circulating LH on the afternoon of proestrus or of midcycle has been associated with an increase in pituitary sensitivity to LH-RH (9-1 1) and also with an increase of LH-RH activity as determined by bioassay (12) or radioimmunoassay in plasma (1 3). Since the administration of an antiserum to synthetic LH-RH suppressed the cyclic surge of LH and FSH in proestrous rats and prevented ovulation (14), we studied the ability of D-Phe2-D-Leu6-LH-RH to suppress the cyclic surge of peripheral LH on the day of proestrus and observed its effects on ovulation in cycling hamsters.