Peptidase activity in the hypothalamus and pituitary of the rat: fluctuations and possible regulatory role of luteinizing hormone releasing hormone-degrading activity during the estrous cycle

Peptidase activity capable of inactivating luteinizing hormone (LHRH) may have a physiological role in partially determining hypothalamic LHRH levels as well as LHRH levels at the gonadotrope. In our previous work ( Lapp and O' Conner , 1984, companion paper), use of the synthetic substrate leucine-p-nitroanilide (Leu-p-NA) to assay LHRH-degradative activity was validated by several methods. The current studies were conducted in order to monitor peptidase activity in the hypothalamus and pituitary throughout the rat 4-day estrous cycle. Activity in both tissues was significantly decreased during proestrus and diestrus I. It seems possible that the proestrous reduction in peptidase activity represents a permissive period necessary for the induction of the LHRH and LH surges. The decreased degradative activity in the pituitary on diestrus I may be involved in inducing the pituitary LHRH receptors which are reportedly synthesized prior to proestrus. The peptidase exhibits positive cooperativity with Leu-p-NA, and the degree of this cooperativity also fluctuates during the estrous cycle. Estradiol and progesterone given alone or in combination to prepubertal castrate animals increased the activity of the hypothalamic peptidase in vitro. The degree of positive cooperativity with which the enzyme functioned was also apparently altered by these gonadal steroids.     

Stimulation of ornithine decarboxylase activity by luteinizing hormone releasing hormone in the testis of immature rat

The activity of ornithine decarboxylase (ODC) was found to increase in the testis of immature rats following intratesticular injection with luteinizing hormone releasing hormone (LHRH). Maximal stimulation of ODC activity occurred with 1 μg of the hormone at 2 h. The enzyme activity returned to control levels at 4 h. The minimal effective dose was found to be 0.1 μg per testis. The stimulating effect of LHRH was confined to Leydig cells alone. The seminiferous tubules did not show any change in ODC activity following LHRH treatment. These results show that LHRH acts directly on the testis and influences the levels of ODC in the Leydig cells of rat.     

Active immunization to luteinizing hormone releasing hormone to inhibit the induction of mammary tumors in the rat

Immunization of female rats with a bovine serum albumin-luteinizing hormone releasing hormone conjugate results in suppression of dimethylbenzanthracene mammary tumor incidence. Tumor incidence was 1.3, and 1.29 tumors per rat in bovine serum albumin alone (n = 10) and unimmunized (n = 18) control groups, but no tumors were found in the bovine serum albumin-luteinizing hormone releasing hormone conjugate immunized animals (n = 10). In a second experiment immunization with bovine serum albumin-luteinizing hormone releasing hormone conjugates reduced tumor incidence to 0.3 tumors per rat (n = 10) from the 1.2 tumors per animal seen in the control animals (n = 10) immunized with bovine serum albumin alone. Bovine serum albumin-luteinizing hormone immunization caused the production of anti-LHRH antibodies, an interruption of estrous cycles, lowered serum estradiol and progesterone levels, and atrophy of the ovaries and uteri. Immunization BSA-hormone conjugates is a novel anti-tumor strategy.     

Pituitary and ovarian responses to luteinizing hormone releasing hormone in a rat with polycystic ovaries

Female rats injected with a single dose of 2 mg estradiol valerate (EV) develop anovulatory acyclicity characterized by persistent vaginal cornification and the formation of multiple large cystic follicles on the ovaries. In order to determine if these effects of EV are accompanied by changes in ovarian and/or pituitary function, the following studies were conducted. Ovarian androgen production was determined by the measurement at 4, 5 and 6 weeks after EV treatment of circulating dehydroepiandrosterone, androstenedione and testosterone. The capacity of the polycystic ovary to ovulate in response to luteinizing hormone releasing hormone (LHRH) stimulus was assessed. Ovarian histology was examined at the termination of the study (9 weeks after EV treatment). Pituitary function was assessed 9 weeks after the EV treatment by examining the acute changes in plasma luteinizing hormone (LH) concentration in response to a double pulse of LHRH. Plasma concentrations of the androgens were unchanged over the 3-week sampling period and were similar to those found in sesame-oil-treated normal cycling control rats. The ovaries from EV-treated animals were smaller than those of controls and the cystic follicles exhibited marked thecal hypertrophy and attenuation of the granulosa cell layer. The basal plasma LH concentration at 9 weeks after EV treatment were significantly lower than in proestrus controls and plasma concentrations of LH elicited by LHRH pulses was significantly lower than in controls. The relative increase in plasma LH following the LHRH stimulus was, however, greater in the EV-treated animals than in controls. In spite of the diminished LH surge elicited in response to LHRH, the EV-treated animals ovulated as indicated by the presence of fresh corpora lutea on the ovaries. These results indicate that androgens are not responsible for the polycystic ovarian condition in this system and that the polycystic ovary is capable of ovulatory function when appropriately stimulated.     

Ontogeny of luteinizing hormone releasing hormone (LHRH) and somatostatin (SRIF) in the hypothalamus of the sheep

Using the immunoperoxidase method, luteinizing hormone releasing hormone (LHRH) and somatostatin (SRIF) were demonstrated in the hypothalamus of fetal sheep. Both hormones were found in the perikarya at about day 60 of fetal life, i.e., at the end of the first half of pregnancy. Immunoreactive LHRH (irLHRH) perikarya were situated in the vicinity of the organum vasculosum of the lamina terminalis (OVLT), i.e., in the medial preoptic nucleus and in the nucleus of the diagonal band of Broca. They were scattered and generally sparse in these areas. In the earliest stages of fetal life (60, 75, 90 days of gestation) irSRIF perikarya grouped in the ventromedial nucleus and in the lateral preoptic nucleus, were very numerous. In the oldest fetuses (120 and 135 days of gestation) they had disappeared from these nuclei but could be found in some extrahypothalamic regions--the amygdala, septo-olfactory area and sometimes in the anterior periventricular zone of the hypothalamus. Neither irLHRH nor irSRIF material were stored in the nerve terminals of the external layer of the median eminence (ME) before day 75 of gestation. In all developmental stages examined, irLHRH material in the ME was very scarce whereas irSRIF material very aboundant.     

Immunocytochemistry of luteinizing hormone releasing hormone (LHRH) and gonadotropic hormones in the sheep after anterior deafferentations of the hypothalamus

Summary Using the immunoperoxidase method, the effect of the anterior deafferentations on the (1) LHRH-neuronal system in the hypothalamus and (2) gonadotropic cells in the adenohypophysis of the ewe were investigated. Two kinds of the anterior deafferentations were placed in the hypothalamus of cycling ewes. The first was performed at the level of caudal border of the chiasma opticum (CB deafferentation) and separated the medio-basal hypothalamus (MBH) from the anterior hypothalamic area (AHA). The second, was placed above the midline of the optic chiasma (MB deafferentation) and detached the AHA from the area praeoptica (AP). Estrous cycles and ovulation ceased in all CB-deafferentation. Immunocytochemical observations revealed a complete lack of LHRH-material both in the hypothalamic nuclei and in all parts of the median eminence (ME) and disappearance of LH-cells in the pituitary gland. In MB deafferented animals, only a diminished density of LHRH-material occurred in the rostral and central parts of the ME, but the ewes continued estrous cycles. Furthermore, numerous LHRH-axons and some LHRH-perikarya were visible in the regions of the AP and AHA. From these results the author is of the opinion, that in the ewe, principally AHA, but not MBH, retains the ability to produce LHRH. Difficulties in staining LHRH-perikarya suggest that in this species LHRH may be synthesized in an immunologically inactive (prohormonal) form.     

Studies on the enzymic degradation of luteinizing hormone releasing hormone by rat pituitary plasma membranes

Degradation of luteinizing hormone releasing hormone (LH-RH) by purified plasma membranes from rat pituitaries was investigated. Synthetic LH-RH (0.5 mg/ml) was incubated (20 min, 37°C) with pituitary plasma membranes (750 μg protein/ml). The reaction was stopped by centrifugation at 4°C. The degradation products were isolated by high pressure liquid chromatography using a reversed-phase column. Amino acid analysis of the degradation products indicated that the N-terminal tripeptide (pGlu-His-Trp) and the N-terminal hexapeptide (pGlu-His-Trp-Ser-Tyr-Gly) sequence of LH-RH are the main degradation products. These results suggest that the main cleavage sites of LH-RH by the pituitary plasma membrane-bound enzymes are the Gly⁶-Leu⁷ and the Trp³-Ser⁴ bonds of the neurohormone.     

Effects of luteinizing hormone releasing hormone on gonadotrophins in the hamster

The changes in serum gonadotrophins in male hamsters following one injection of 15 μg luteinizing hormone releasing hormone (LHRH) (Group A) were compared with those following the last injection of LHRH in animals receiving an injection approximately every 12 hr for 4 days (Group B) or 12 days (Group C). Peak follicle stimulating hormone (FSH) levels (ng/ml) were 1776±218 (Group A), 2904±346 (Group B), and 4336±449 (Group C). Peak luteinizing hormone (LH) values (ng/ml) were 1352±80 (Group A), 410±12 (Group B), and 498±53 (Group C). Serum FSH:LH ratios, calculated from the concentrations measured 16 hr after the last LHRH injections, were higher in Groups B and C than in Group A. Similar injections of LHRH (100 ng or 15 μg/injection) for 6 days elevated the serum FSH:LH ratio in intact males. Five such LHRH injections (100 ng/injection) blunted the rise in serum LH in orchidectomized hamsters. Direct effects of LHRH on gonadotrophin secretory dynamics or altered brain-pituitary-testicular interactions may alter the ratio of FSH to LH in the hamster.     

Some analogs of luteinizing hormone releasing hormone (LH-RH) having intense ovulation-inducing activity

Five new analogs of luteinizing hormone releasing hormone (LH-RH), des-Gly¹⁰-[Ala⁶]-LH-RH-ethylamide, des-Gly¹⁰-[D-Ala⁶]-LH-RH-ethylamide, des-Gly¹⁰-[α-aminoisobutyric acid⁶]-LH-RH-ethylamide, des-Gly¹⁰-[Phe⁵, D-Ala⁶]-LH-RH-ethylamide and des-Gly¹⁰-[Ile⁵, D-Ala⁶]-LH-RH-ethylamide were synthesized and evaluated for the ovulation-inducing activity in the rat, and it was found that the analogs, des-Gly¹⁰-[D-Ala⁶]-LH-RH-ethylamide and des-Gly¹⁰-[Phe⁵, D-Ala⁶]-LH-RH-ethylamide, were 50 times or more active than the original molecule.