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.     

Prolactin releasing and luteinizing hormone inhibiting activity of dermorphin shorter homologues in the rat

Dermorphin, a heptapeptide isolated from the skin of the frogs Phillomedusa sauvagei and Phillomedusa rhodei, is endowed with potent peripheral and central opioid-like activity. Intracerebroventricular (icv) injection of dermorphin (31.2, 62.5 and 125 pmol/100g) induced in ovariectomized (OVX) rats dose related rises and decreases in prolactin (PRL) and luteinizing hormone (LH) levels, respectively. The aim of this work was to evaluate the same endocrine responses after administration of shorter peptide amide homologues, related to the N-terminal sequence of dermorphin. These compounds retain a substantial analgesic activity although the latter decreases with the decrease in the number of amino acid residues. Icv administration of the hexapeptide homologue (dermorphin 1-6 amide) to OVX rats did not induce any PRL rise or LH inhibition, even at the high dose of 250 pmol/100g. The pentapeptide (dermorphin 1-5 amide), instead, increased PRL and decreased LH secretion, although the effect was significant only at the dose of 250 pmol/100g. Administration of the tetrapeptide (dermorphin 1-4 amide) induced a significant PRL rise and LH inhibition at both the doses of 125 and 250 pmol/100g. The tetrapeptide was the smallest fragment of the dermorphin moiety which caused endocrine responses while the tripeptide (dermorphin 1-3 amide) was completely ineffective in this context. These data indicate that a complete dissociation exists between the behavioral and endocrine effects of the dermorphin homologues examined. In fact, shorter dermorphins whose analgesic potency was directly related to the number of amino acids, exhibited an opposite pattern in evoking endocrine effects.     

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.     

Immunocytochemistry of luteinizing hormone releasing hormone (LHRH) in the sheep hypothalamus during various reproductive stages

Summary Using the labeled and unlabeled immunoperoxidase methods, the distribution and concentration (1) of immunoreactive LHRH-material in the hypothalamus, and (2) of gonadotropic hormones in the adenohypophysis of the ewe were determined during various reproductive stages, including two phases of the estrous cycle, anestrus, lactation, and the state after ovariectomy. The concentration of LHRH-immunoreactive material varied in particular regions of the median eminence (ME) and was closely dependent on the physiological state. The immunoreactive material was most abundant in hypothalami of lactating animals, exceeding gradually the corresponding deposits in ovariectomized animals, in ewes during the 16th day of the estrous cycle (before ovulation), and in anestrous ewes. A severe depletion of LHRH from the rostral and central parts of the ME was observed 24h after ovulation. This depletion was accompanied by a degranulation of LH-and prolactin-producing cells in the adenohypophysis. It is postulated that LHRH is stored in the ME of the sheep in all examined endocrine stages. Only circumscribed regions of the ME take part in the cyclic release of LHRH during the ovulatory phase; this suggests a functional differentiation of the nerve terminals in this neurohemal area.     

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.     

Subcellular distribution of the enzymes degrading thyrotropin releasing hormone and metabolites in rat brain

In order to further understand the role of enzymes degrading Thyrotropin Releasing Hormone (TRH, pglu-his-proNH₂) and metabolites, we studied their subcellular distribution in rat brain. Brain tissue was homogenized in 0.32 M sucrose, tris-HCl 0.01 M pH 7.4 and fractionated by differential and discontinuous gradient centrifugation; [³H]pro-TRH was incubated with the various subcellular fractions and the extent of degradation of each metabolite was measured after separation by thin layer chromatography. Several markers were simultaneously measured (lactate dehydrogenase, 5′-nucleotidase and hexosaminidase) to determine the pattern of distribution of the subcellular organelles. The post-proline cleaving enzyme responsible for pglu-his-pro formation and pyroglutamate amino-peptidase (which requires sulphydryl compounds for maximal activity) were found in cytosol but were barely detectable in the soluble component of synaptosomes; pyroglutamate aminopeptidase (dependent on metals) and post-proline dipeptidyl amino peptidase were found on the membranes of synaptosomes; imido peptidase was not enriched in any particular fraction.These data are consistent with the hypothesis that membrane-bound pyroglutamate aminopeptidase is responsible for TRH degradation once released into the synaptic cleft and that the post-proline dipeptidylaminopeptidase may participate in the extracellular catabolism of his-proNH₂ before it cyclizes to his-pro-DKP. They also suggest that post-proline cleaving enzyme and soluble pyroglutamate aminopeptidase may not play an important role in the regulation of TRH levels in nerve endings.     

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.