GnRH is a ten amino acid peptide originally isolated from the hypothalamus and is responsible for the release of both LH and FSH. Adelman et al. (1986) isolated genomic and cDNA encoding the precursor of GnRH. These DNA sequences code for a protein of 92 amino acids in which the GnRH decapeptide is preceded by a signal peptide of 23 amino acids and followed by a gly-lys-arg sequence as expected for enzymatic cleavage of the decapeptide from its precursor and amidation of the carboxyterminal of GnRH.
GnRH and its agonists stimulate oocyte meiosis and ovulation in hypophysectomized rats and in vitro in follicle-enclosed oocytes (Ekholm et al., 1981 and Hillensjo et al., 1980). Gonadotropin-releasing hormone (GnRH) and its agonistic analogs inhibited follicle-stimulating hormone (FSH)-induced increase of estrogen and progesterone production in vitro by rat ovarian granulosa cells (Hsueh et al., 1979 and 1981). These peptides also induce follicle atresia (Billig et al., 1994). A GnRH agonist was shown to Inhibit human granulosa cell progesterone secretion (Tureck et al., 1982).
Acute depletion of murine primordial follicle reserve by gonadotropin-releasing hormone antagonists Danforth DR,et al .
OBJECTIVE: To examine the effects of GnRH antagonists on preantral follicle survival in vivo and to investigate whether GnRH antagonist use during cyclophosphamide treatment would protect the ovary and preserve primordial follicle survival in a murine model. DESIGN: Prospective basic research study. SETTING: Research laboratory in an academic medical center. ANIMAL(S): Adult C57Bl/6 mice (5 to 6 weeks old). INTERVENTION(S): Mice received either a single injection of GnRH agonist (leuprolide acetate) on study day -10 or injections of the GnRH antagonist (antide or cetrorelix) on study days -3 and 0. Some animals also received the chemotherapeutic agent cyclophosphamide on day 0. All animals were killed by CO(2) asphyxiation on day 7. To examine direct vs. indirect effects, some mice received GnRH antagonist under the bursa of one ovary, with the contralateral ovary receiving vehicle. Ovaries were fixed in Kahle's solution; 7-mum tissue sections were stained with Lillie's allochrome, and preantral follicles were counted on every fifth section. MAIN OUTCOME MEASURE(S): Numbers of primordial, primary, and secondary follicles. RESULT(S): Systemic administration of both GnRH antagonists caused a significant destruction of primordial follicles compared with control mice. Similar results were obtained whether the antagonists were administered systemically or directly to the ovary. Gonadotropin-releasing hormone agonist had no effect on primordial follicle numbers by itself but reduced the follicular depletion caused by cyclophosphamide. CONCLUSION(S): In contrast to the effects of GnRH agonists to reduce chemotherapeutic destruction of primordial follicles, GnRH antagonists do not protect the ovary from the damaging effects of cyclophosphamide. More importantly, GnRH antagonists alone deplete primordial follicles in this murine model, likely through a direct effect on the ovary. Whether these observations apply to other species requires further study.
Effects of gonadotrophin-releasing hormone agonists on apoptosis of granulosa cells Tsai NM, et al .
Granulosa cells are known to contribute to maturation of oocytes, and most of the growth factors exert their action via granulosa cells. It has been established that granulosa cell death during follicular atresia and luteolysis results from apoptosis. However, the precise mechanistic pathways of granulosa cell apoptosis have not yet been defined. In this study, we determined the proportions of apoptosis in granulosa cells treated with two kinds of gonadotrophin-releasing hormone agonists (GnRHa): buserelin and leuprorelin depot. The incidences of DNA fragmentation of human granulosa cells treated with buserelin and leuprorelin were 54.33% and 39.02%, respectively. The proportions of apoptotic bodies were 6.04% and 4.29%, respectively. There was a significant difference in the proportions of DNA fragmentation between the two kinds of GnRHa-treated granulosa cells. The apoptosis pathway and associated protein expression in granulosa cells treated with GnRHa were also determined. The Bax molecule, a pro-apoptosis protein, was expressed in granulosa cells undergoing apoptosis. In contrast, Bcl-2, an anti-apoptosis protein, could not be detected in the same group of granulosa cells. The distribution of cytochrome c determined via immunostaining showed a diffuse pattern, which most likely indicated that cytochrome c was translocated from mitochondria into the cytoplasm. Western blotting showed the expressions of caspase-9 and caspase-3 in patients' granulosa cells. The GnRHa effects on granulosa cells indicated a higher incidence of DNA fragmentation and apoptotic bodies in the buserelin-treated than in the leuprorelin depot-treated group. The granulosa cells go through the mitochondria-dependent apoptosis pathway; the indicated pro-apoptosis protein Bax was expressed and induced cytochrome c release from mitochondria, which then activated caspase-9 and caspase-3 until cell death occurred.
Expression regulated by
FSH, LH
Comment
Gonadotropin-Releasing Hormone-I or -II Interacts with IGF-I/Akt But Not Connexin 43 in Human Granulosa Cell Apoptosis. Hong IS et al. Background:We have recently demonstrated that GnRH-I or -II can induce apoptosis in immortalized human granulosa cells by activating the caspase signaling cascade. Whether GnRH-I or -II can affect other regulators such as Bcl-2 family members, IGF-I, or gap junctions and the mechanisms involved are unknown.Methods:Immortalized human granulosa cells were treated with GnRH-I, GnRH-II, IGF-I, or antide (a GnRH-I receptor antagonist), in various combinations. Cell proliferation and apoptotic changes were evaluated by cell counting, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and immunoblotting. Activated or total protein expression of IGF-I receptor, Akt, connexin 43 (Cx43), or caspase-3 with and without dominant-negative Akt (an Akt-suppressing vector), wortmannin (a phosphatidylinositol-3-kinase inhibitor), or Cx43 small interfering RNA transfection were assessed by immunoblotting. Gap junctional communication was determined by dye transfer assay.Results:GnRH-I or -II inhibited cell proliferation, induced TUNEL-positive cells, and increased caspase-3 activities but had no effects on Bcl-2 family members. IGF-I increased cell proliferation, decreased TUNEL-positive cells and caspase-3 activities, and increased Akt activities, and these effects were attenuated by GnRH-I or -II. Effects of IGF-I on caspase-3 activities were attenuated by dominant-negative Akt or wortmannin. GnRH-I or -II decreased dye transfer, increased Cx43 phosphorylation, and increased caspases-3 activities even after Cx43 knockdown.Conclusion:GnRH-I or -II induces apoptosis in human granulosa cells through a caspase-3-dependent extrinsic pathway rather than a Bcl-2 family-dependent intrinsic pathway and attenuates the antiapoptotic action of IGF-I through Akt. Cx43-induced gap junctional changes do not initiate granulosa cell apoptosis but likely result from apoptosis induced by GnRH-I or -II.
Kang SK, et al 2000 reported differential Regulation of Two Forms of Gonadotropin-Releasing
Hormone Messenger Ribonucleic Acid in Human
Granulosa-Luteal Cells.
The recent cloning of a second form of
GnRH (GnRH-II) with characteristics of chicken GnRH-II in the primate brain has
prompted a reevaluation of the role of GnRH in reproductive functions. The authors investigated the hormonal regulation of GnRH-II messenger
RNA (mRNA) and its functional role in the human granulosa-luteal cells (hGLCs),
and provided evidence for differential hormonal regulation of GnRH-II
vs. GnRH-I mRNA expression.
The expression levels of GnRH-II, GnRH-I, and GnRH receptor (GnRHR) mRNA
were investigated using semiquantitative or competitive RT-PCR. A significant
decrease in GnRH-II and GnRHR mRNA levels was observed in cells treated
with GnRH-II or GnRH-II-a. In contrast, GnRH-I-a revealed a biphasic effect (up-
and down-regulation) of GnRH-I and GnRHR mRNA, suggesting that GnRH-I and
GnRH-II may differentially regulate GnRHR and their ligands (GnRH-I and
GnRH-II). Treatment with FSH or hCG increased GnRH-II mRNA levels but
decreased GnRH-I mRNA levels, further indicating that GnRH-I and GnRH-II
mRNA levels are differentially regulated. To investigate the physiological role of
GnRH-II, hGLCs were treated with GnRH-II or GnRH-II-a in the presence or
absence of hCG, for 24 h, and progesterone secretion was measured by RIA. Both
GnRH-II and GnRH-II-a inhibited basal and hCG-stimulated progesterone
secretion, effects which were similar to the effects of GnRH-I treatment on
ovarian steroidogenesis. Next, hGLCs were treated with various concentrations of
GnRH-II, GnRH-II-a, or GnRH-I-a; and the expression levels of FSH receptor and
LH receptor were investigated using semiquantitative RT-PCR. A significant
down-regulation of FSH receptor and LH receptor was observed in cells treated
with GnRH-II, GnRH-II-a, and GnRH-I-a, demonstrating that GnRH-II and
GnRH-I may exert their antigonadotropic effect by down-regulating gonadotropin
receptors. Interestingly, GnRH-II and GnRH-II-a did not affect basal and
hCG-stimulated intracellular cAMP accumulation, suggesting that the
antigonadotropic effect of GnRH-II may be independent of modulation of cAMP
levels. Taken together, these results suggest that GnRH-II may have biological
effects similar to those of GnRH-I but is under differential hormonal regulation in
the human ovary.
Ovarian localization
Granulosa
Comment
Oikawa et al. (1990) demonstrated the expression of GnRH mRNA in rat granulosa cells using reverse transcription-PCR.
Immunolocalization of Gonadotropin-Releasing Hormone (GnRH)-I, GnRH-II, and Type-I GnRH Receptor during Follicular Development in the Human Ovary. Choi JH et al. Context: Gonadotropin releasing hormone (GnRH) and its receptor have been detected, at the mRNA level, in different ovarian cell types, implicating an autocrine role of the GnRH system in the human ovary. However, the expression, at the protein level, of GnRH and its receptor in specific cell types during follicular development has not been documented in humans. Objective: We evaluated the immunohistochemical expression of GnRH-I (the classical form of mammalian GnRH), GnRH-II (the novel isoform) and the type-I GnRH receptor (GnRHR) that is known to bind both forms of GnRH, in ovaries of pre-menopausal women. Main Outcome Measures: Immunohistochemistry, immunofluorescence, immunoblot assay and real-time RT-PCR were performed. Results: GnRH-I, GnRH-II and GnRHR were not immunostained in the follicles from the primordial to the early antral stage. In preovulatory follicles, both forms of GnRH and their common receptor were localized predominantly to the granulosa cell layer, whereas the theca interna layer was weakly positive. In the corpus luteum, significant levels of GnRH-I, GnRH-II as well as GnRHR were observed in granulosa luteal cells, but not in theca luteal cells. Both GnRH isoforms and the type-I GnRHR were localized also to the ovarian surface epithelium (OSE) from which over 85% of ovarian cancer are thought to be derived. Conclusion: The expression of GnRH-I, GnRH-II and GnRHR protein in the human ovary is temporally and spatially specific, and further support the physiological role of an autocrine regulatory system involving GnRH-I, GnRH-II and GnRHR in follicular development and corpus luteal function.
Follicle stages
Antral, Preovulatory, Corpus luteum
Comment
Gonadotropin-Releasing Hormone 1 Directly Affects Corpora Lutea Life-Span in Mediterranean Buffalo (Bubalus bubalis) During Diestrus: Presence and In Vitro Effects on Enzymatic and Hormonal Activities. Zerani M et al. The expression of gonadotropin-releasing hormone (GNRH) receptor (GNRHR) and the direct role of GNRH1 on corpora lutea function were studied in Mediterranean buffalo during diestrus. Immunohistochemistry evidenced at early, mid, and late luteal stages the presence of GNRHR only in large luteal cells and GNRH1 in both small and large luteal cells. Real-time-PCR revealed GNRHR and GNRH1 mRNA at the three luteal stages, with lowest values in late corpora lutea. In vitro corpora lutea progesterone production was greater in mid stages and less in late luteal phases, while prostaglandin (PG) F2a (PGF2a) increased from early to late stages, and PGE2 was greater in the earlier-luteal phase. Cyclooxygenase 1 (prostaglandin-endoperoxide synthase 1, PTGS1) activity did not change during diestrus, while PTGS2 increased from early to late stages, and PGE2-9-ketoreductase (PGE2-9-K) was greater in late corpora lutea. PTGS1 activity was greater than PTGS2 in early corpora lutea and less in late luteal phase. In corpora lutea cultured in vitro, the GNRH1 analog (buserelin) reduced progesterone secretion, and increased PGF2a secretion as well as PTGS2 and PGE2-9-K activities at mid and late stages. PGE2 release and PTGS1 activity were increased by buserelin only in late corpora lutea. These results suggest that GNRH is expressed in all luteal cells of buffalo, whereas GNRHR only in large luteal phase. Additionally, GNRH directly down-regulates corpora lutea progesterone release, with the concomitant increases of PGF2a production and PTGS2 and PGE2-9-K enzymatic activities.
GnRH actions on rat preovulatory follicles are mediated by paracrine EGF-like factors. Motola S et al. Gonadotropin releasing hormone (GnRH) has been shown to mimic the actions of LH/hCG on oocyte maturation and ovulation. Recent studies demonstrated that induction of ovulation by LH/hCG is mediated, at least in part, by transactivation of epidermal growth factor receptors (EGFR) by autocrine/paracrine EGF-like factors activated by metalloproteases. Here we have examined whether the action of GnRH on the preovulatory follicles is exerted through similar mechanisms involving activation of EGFR. The EGFR kinase inhibitor, AG1478, inhibited GnRH-induced oocyte maturation in explanted follicles in vitro. Its inactive analog, AG43, did not affect GnRH-stimulated resumption of meiosis. GnRH, like LH, stimulated transient follicular expression of EGF-like agents, as well as rat cycloxygenase-2 (rCOX-2), rat hyaluronan synthase-2 (rHAS-2), and rat tumor necrosis factor-alpha-stimulated gene 6 (rTSG-6) mRNAs, known ovulatory enzymes. Likewise, GnRH stimulated follicular progesterone synthesis. Conversely AG1478 inhibited all these actions of GnRH. Furthermore, Galardin, a broad-spectrum metalloprotease inhibitor, blocked GnRH-induced oocyte maturation and follicular progesterone synthesis. In conclusion, we have demonstrated that follicular EGF-like factors mediate also the GnRH-stimulation of ovulatory changes, like these of LH/hCG. Mol. Reprod. Dev. (c) 2006 Wiley-Liss, Inc.
Phenotypes
Mutations
1 mutations
Species: mouse
Mutation name: hpg
type: naturally occurring fertility: None Comment: Hereditary hypogonadism in the hypogonadal (hpg)
mouse is caused by a deletional mutation of at least 33.5
kilobases encompassing the distal half of the gene for
the common biosynthetic precursor of
gonadotropin-releasing hormone (Mason et al., 1986).