Yitzhak Koch

Gonadotropin Action on Cultured Graafian Follicles: Induction of Maturation Division of the Mammalian Oocyte and Differentiation of the Luteal Cell

Publisher Summary This chapter discusses the nature of interactions between oocyte and granulose cells and the triggering effect of luteinizing hormone (LH). The mammalian oocyte embarks on its first reduction division in prenatal life or during the early postnatal period. This division has a protracted and complicated prophase. Just before or shortly after birth, depending on the species, the germ cell reaches the stage of diplotene. By this time, the oocyte has doubled its DNA complement, the chromosomes have condensed, and homologous chromosomes have become paired and are linked by chiasmata permitting the exchange of paternal and maternal genetic information. In murid rodents, the chromosomes decondense and resume their transcriptive activity. In the adult, during each estrous cycle, a number of oocytes characteristic of the species complete their first reduction division, resulting in the abstriction of the first polar body shortly before ovulation. This resumption of meiosis and its progress to the metaphase of the second meiotic division refer to as ovum maturation. Completion of the second meiotic division, with extrusion of a second polar body, occurs only upon penetration of the oocyte by a spermatozoon.

Ovulation rate and serum LH levels in rats treated with indomethacin or prostaglandin E2.

Abstract Serum LH levels were determined by radioimmunoassay at the normal time of the proestrous LH peak (17.30 – 18.00) and ovulatory performance was examined on the morning of estrus in rats treated with indomethacin, an inhibitor of prostaglandin synthesis. When the drug was administered at 14.30 on the day of proestrus, only 21% of the rats ovulated and the total number of ova shed was reduced to 4% of that found in the untreated control group, but there was no significant change in peak serum LH level (1122 ± 184 vs. 975 ± 240 ng/ml ± S.E., treated vs. control). Prostaglandin E2 (PGE2) given late on the day of proestrus (25 to 750 μ g/rat at 24.00) was effective in overcoming this antiovulatory action of indomethacin: 71–90% of the rats ovulated, though the number of eggs shed was low (24–55% of control value). Indomethacin was still effective in blocking ovulation when given at 20.00, that is after completion of the proestrous LH surge, but not at 24.00. Administration of PGE2 (2 × 750 μ g/rat) in the early afternoon of proestrus elicited a rise in serum LH levels in rats in which the cyclic LH surge had been blocked with Nembutal (470 ± 87 vs. 106 ± 17 ng/ml ± S.E.) and induced ovulation in two-thirds of these animals. The results confirm, by direct measurement, that indomethacin does not block LH release but interferes with a late phase of the ovulatory process. PGE2 reverses this action of indomethacin on the ovary. In addition, PGE2 has a central effect causing LH release.

A second isoform of gonadotropin-releasing hormone is present in the brain of human and rodents

Gonadotropin‐releasing hormone‐I (GnRH‐I), present in the mammalian hypothalamus, regulates reproduction. In this study we demonstrate, for the first time, that an additional isoform of GnRH, [His5, Trp7, Tyr8] GnRH‐I (GnRH‐II) is present in the brain of the mouse, rat and human. Human and rat brain extracts contain two isoforms of GnRH, GnRH‐I and GnRH‐II, which exhibited identical chromatographic properties to the respective synthetic peptides, in high performance liquid chromatography. Using immunohistochemical techniques we have found that GnRH‐II is present in neuronal cells that are localized mainly in the periaqueductal area as well as in the oculomotor and red nuclei of the midbrain. It is of interest to note that in the hypogonadal mouse, although the GnRH‐I gene is deleted, GnRH‐II is present. Substantial concentrations of GnRH‐II are also present in the hypothalamus and stored in the human pituitary stalk or in the mouse median eminence. By using reverse transcription (RT)‐PCR we have also found that while GnRH‐II is not expressed in the cerebellum, it is expressed in all three structures of the brain stem: midbrain, pons and medulla oblongata.

Production and characterization of an antiserum to synthetic gonadotropin-releasing hormone.

Abstract The synthetic decapeptide “luteinizing hormone-releasing hormone” (LH-RH) was rendered antigenic by reaction of its histidine or tyrosine residues (7 : 3 approx.) with p-diazonium phenylacetic acid and coupling of the azo-derivatives formed to bovine serum albumin (BSA). Immunization of rabbits yielded antisera that bound 125 I-labeled LH-RH (approx. 50 pg) at dilutions up to 1:200, 000 and showed no cross-reaction with unrelated hypothalamic and pituitary hormones, extracts from rat cerebral cortex, and with small fragments of LH-RH. Cross-reaction was minimal (0.2%) with the free acid analogue of LH-RH, and moderate with des -pGlu LH-RH (20%), des -pGlu-His-LH-RH (2.4%) and with LH-RH analogues in which a single residue (No. 4–6 or No. 8) was exchanged by an amino-acid of similar character (1.2–12%). Biologically active hypothalamic extract and LH-RH produced parallel 125 I-LH-RH-binding inhibition curves, providing immunochemical support for the identity of the native releasing hormone with synthetic LH-RH.

The neuropeptides GnRH-II and GnRH-I are produced by human T cells and trigger laminin receptor gene expression, adhesion, chemotaxis and homing to specific organs

Can T cells be directly activated to de novo gene expression by gonadotropin-releasing hormone-II (GnRH-II), a unique 10-amino-acid neuropeptide conserved through 500 million years of evolution? GnRH-II, which has been identified in mammals, shares 70% homology with the mammalian hypothalamic neurohormone GnRH (GnRH-I), the primary regulator of reproduction, but is encoded by a different gene. Although both neuropeptides are produced mainly in brain, their localization and promoter regulation differ, suggestive of distinct functions. Indeed, GnRH-II barely affects reproduction and its role in mammalian physiology is unknown. We find here that human normal and leukemic T cells produce GnRH-II and GnRH-I. Further, exposure of normal or cancerous human or mouse T cells to GnRH-II or GnRH-I triggered de novo gene transcription and cell-surface expression of a 67-kD non-integrin laminin receptor that is involved in cellular adhesion and migration and in tumor invasion and metastasis. GnRH-II or GnRH-I also induced adhesion to laminin and chemotaxis toward SDF-1α, and augmented entry in vivo of metastatic T-lymphoma into the spleen and bone marrow. Homing of normal T cells into specific organs was reduced in mice lacking GnRH-I. A specific GnRH-I-receptor antagonist blocked GnRH-I- but not GnRH-II-induced effects, which is suggestive of signaling through distinct receptors. We suggest that GnRH-II and GnRH-I, secreted from nerves or autocrine or paracrine sources, interact directly with T cells and trigger gene transcription, adhesion, chemotaxis and homing to specific organs, which may be of clinical relevance.

Enzymic degradation of luteinizing hormone-releasing hormone (LH-RH) by hypothalamic tissue

Abstract Synthetic luteinizing hormone-releasing hormone (LH-RH) lost both its immunore-activity and hormonal activity on incubation with hypothalamic or cerebrocortical slices or homogenates. This inactivation was shown to be due to degradation of the decapeptide by soluble enzyme(s) present in the 100,000 × g supernatant fraction of the homogenates. The supernatant derived from one rat hypothalamus was capable of destroying 1 μg of exogenous LH-RH within 5 min. The hexapeptide pGlu-His-Trp-Ser-Tyr-Gly was identified as the major radioactive breakdown product of [pGlu-3-3H] LH-RH, and tentative evidence for the formation of the tetrapeptide Leu-Arg-Pro-Gly-NH2 was obtained by sequential electrophoresis and paper chromatography. These findings suggest that the Gly-Leu bond may be the preferred site of cleavage.

Resistance to enzymic degradation of LH-RH analogues possessing increased biological activity

Abstract Three analogues of LH-RH in which Dextrarotatory amino acids were substituted for the Gly 6 , and two additional analogues in which the Leu 7 residue was also modified, were subjected to enzymic preparations derived from rat hypothalamus or anterior pituitary. These enzymes, known to cleave LH-RH, preferentially at the Gly 6 -Leu 7 position, proved less effective in degrading all the analogues tested. Among the Gly 6 substituted analogues, [D-Trp 6 ] LH-RH, having the highest LH-releasing activity, was most resistant to degradation. Additional modification, at position 7, although rendering the analogues immune to enzymic attack, did not further enhance their biological potency. These data suggest that degradation of LH-RH is a physiological determinant of its biological activity and has therefore to be considered with on designing new, potent analogues of the hormone.

The gonadotropin‐releasing hormone family of neuropeptides in the brain of human, bovine and rat: identification of a third isoform

The mammalian gonadotropin‐releasing hormone (GnRH‐I), which regulates reproduction, was the first isoform of GnRH that was identified in mammals. Recently, we and others have demonstrated the existence of a second isoform of GnRH in the brain of mammals. The presence of a third isoform of GnRH, GnRH‐III, in the brain of mammals is reported herein. GnRH‐III, extracted from the brain of bovine and human, was purified by high performance liquid chromatography, using two distinct elution programs. In both, GnRH‐III was eluted at the same positions as synthetic salmon GnRH, as demonstrated by radioimmunoassay. The luteinizing hormone‐releasing activity of purified GnRH‐III, using dispersed rat pituitary cells, was found to be similar to that of synthetic salmon GnRH. The total amount of GnRH‐III, determined by radioimmunoassay, in the hypothalamus and midbrain of humans and calves is similar to that of GnRH‐I. Immunohistochemical studies demonstrated GnRH‐III‐containing neurons in the hypothalamus and midbrain of human and GnRH‐III fibers in the median eminence of rats. The distribution of GnRH‐III in the brain suggests that in addition to a putative function as a neurohormone at the hypothalamic–pituitary axis, GnRH‐III may have other functions. Our present results suggest that multiple isoforms of GnRH are present in the brain of mammals, and further studies are required in order to elucidate their biological functions.

Suppression of gonadotropin secretion and prevention of ovulation in the rat by antiserum to synthetic gonadotropin-releasing hormone

Abstract Administration of an antiserum (0.10–0.25 ml/rat) to the synthetic decapeptide “luteinizing hormone releasing hormone” (LH-RH) suppressed the cyclic surge of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in proestrous rats and prevented ovulation; exogenous LH reversed the block of ovulation. Serum prolactin levels remained unaffected. In ovariectomized rats, the antiserum suppressed the elevated serum levels of both gonadotropins. These findings are compatible with the view that the synthetic decapeptide is identical with the natural hypothalamic hormone that regulates the secretion of both LH and FSH.

Release of thyrotropin releasing hormone into hypophysial portal blood is high relative to other neuropeptides and may be related to prolactin secretion

The amount of immunoreactive TRH released into hypophysial portal blood of female rats was about 2 orders of magnitude greater than gonadotropin releasing hormone and somatostatin. The turnover of TRH, as high as 80% of the total hypothalamic content per hour, was also much greater than that of any other known peptide. TRH release increased during the expected proestrous surge of prolactin and also in some animals during suckling.