Skip to main content

Advertisement

Log in

Immunohistochemical localization of GnRH and RFamide-related peptide-3 in the ovaries of mice during the estrous cycle

  • Original Paper
  • Published:
Journal of Molecular Histology Aims and scope Submit manuscript

Abstract

Gonadotropin releasing hormone (GnRH) has now been suggested as an important intraovarian regulatory factor. Gonadotropin inhibitory hormone (GnIH) a hypothalamic dodecapeptide, acts opposite to GnRH. GnRH, GnIH and their receptors have been demonstrated in the gonads. In order to find out the physiological significance of these neuropeptides in the ovary, we aim to investigate changes in the abundance of GnRH I and GnIH in the ovary of mice during estrous cycle. The present study investigated the changes in GnRH I, GnRH I-receptor and RFRP-3 protein expression in the ovary of mice during estrous cycle by immunohistochemistry and immunoblot analysis. The immunoreactivity of GnRH I and its receptor and RFRP-3 were mainly localized in the granulosa cells of the healthy and antral follicles during proestrus and estrus and in the luteal cells during diestrus 1 and 2 phases. The relative abundance of immunoreactivity of GnRH I, GnRH I-receptor and RFRP-3 undergo significant variation during proestrus and thus may be responsible for selection of follicle for growth and atresia. A significant increase in the concentration of RFRP-3 during late diestrus 2 coincided with the decline in corpus luteum activity and initiation of follicular growth and selection. In general, immunolocalization of GnRH I, GnRH I-receptor and RFRP-3 were found in close vicinity suggesting functional interaction between these peptides. It is thus, hypothesized that interaction between GnRH I-RFRP-3 neuropeptides may be involved in the regulation of follicular development and atresia.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  • Andreu C, Parborell F, Vanzulli S, Chemes H, Tesone M (1998) Regulation of follicular luteinization by a gonadotropin-releasing hormone agonist: relationship between steroidogenesis and apoptosis. Mol Reprod Dev 51:287–294

    Article  PubMed  CAS  Google Scholar 

  • Aten RF, Williams AT, Behrman HR (1986) Ovarian gonadotropin-releasing hormone-like protein(s): demonstration and characterization. Endocrinology 118:961–967

    Article  PubMed  CAS  Google Scholar 

  • Bentley GE, Kriegsfeld LJ, Osugi T, Ukena K, O’Brien S, Perfito N, Moore IT, Tsutsui K, Wingfield JC (2006) Interactions of gonadotropin-releasing hormone (GnRH) and gonadotropin-inhibitory hormone (RFRP-3) in birds and mammals. J Exp Zool A Comp Exp Biol 305:807–814

    PubMed  Google Scholar 

  • Bentley GE, Ubuka T, McGuire NL, Chowdhury VS, Morita Y, Yano T, Hasunuma I, Binns M, Wingfield JC, Tsutsui K (2008) Gonadotropin-inhibitory hormone and its receptor in the avian reproductive system. Gen Comp Endocrinol 156:34–43

    Article  PubMed  CAS  Google Scholar 

  • Billig H, Furuta I, Hsueh AJ (1994) Gonadotropin-releasing hormone directly induces apoptotic cell death in the rat ovary: biochemical and in situ detection of deoxyribonucleic acid fragmentation in granulosa cells. Endocrinology 134:245–252

    Article  PubMed  CAS  Google Scholar 

  • Birnbaumer L, Shahabi N, Rivier J, Vale W (1985) Evidence for a physiological role of gonadotropin-releasing hormone (GnRH) or GnRH-like material in the ovary. Endocrinology 116:1367–1370

    Article  PubMed  CAS  Google Scholar 

  • Brus L, Lambalk CB, de Koning J, Helder MN, Janssens RM, Schoemaker J (1997) Specific gonadotrophin-releasing hormone analogue binding predominantly in human luteinized follicular aspirates and not in human pre-ovulatory follicles. Hum Reprod 12:769–773

    Article  PubMed  CAS  Google Scholar 

  • Chakrabarti N, Subbarao T, Sengupta A, Xu F, Stouffer RL, Sridaran R (2008) Expression of mRNA and proteins for GnRH I and II and their receptors in primate corpus luteum during menstrual cycle. Mol Reprod Dev 75:1567–1577

    Article  PubMed  CAS  Google Scholar 

  • Erickson GF, Li D, Sadrkhanloo R, Liu XJ, Shimasaki S, Ling N (1994) Extrapituitary actions of gonadotropin-releasing hormone: stimulation of insulin-like growth factor-binding protein-4 and atresia. Endocrinology 134:1365–1372

    Article  PubMed  CAS  Google Scholar 

  • Gibson EM, Humber SA, Jain S, Williams WP, Zhao S, Bentley GE, Tsutsui K, Kriegsfeld LJ (2008) Alterations in RFamide-related peptide expression are coordinated with the preovulatory luteinizing hormone surge. Endocrinology 149:4958–4969

    Article  PubMed  CAS  Google Scholar 

  • Hillensjo T, LeMaire WJ (1980) Gonadotropin releasing hormone agonists stimulate meiotic maturation of follicle-enclosed rat oocytes in vitro. Nature 287:145–146

    Article  PubMed  CAS  Google Scholar 

  • Hsueh AJ, Schaeffer JM (1985) Gonadotropin-releasing hormone as a paracrine hormone and neurotransmitter in extra-pituitary sites. J Steroid Biochem Mol Biol 23:757–764

    CAS  Google Scholar 

  • Jones PB, Hsueh AJ (1981) Regulation of ovarian 20 alpha-hydroxysteroid dehydrogenase by gonadotropin releasing hormone and its antagonist in vitro and in vivo. J Steroid Biochem Mol Biol 14:1169–1175

    CAS  Google Scholar 

  • Jones PB, Conn PM, Marian J, Hsueh AJ (1980) Binding of gonadotropin releasing hormone agonist to rat ovarian granulosa cells. Life Sci 27:2125–2132

    Article  PubMed  CAS  Google Scholar 

  • Knecht M, Ranta T, Feng P, Shinohara O, Catt KJ (1985) Gonadotropin-releasing hormone as a modulator of ovarian function. J Steroid Biochem Mol Biol 23:771–778

    CAS  Google Scholar 

  • Kogo H, Kudo A, Park MK, Mori T, Kawashima S (1995) In situ detection of gonadotropin-releasing hormone (GnRH) receptor mRNA expression in the rat ovarian follicles. J Exp Zool 272:62–68

    Article  PubMed  CAS  Google Scholar 

  • Kriegsfeld LJ, Mei DF, Bentley GE, Ubuka T, Mason AO, Inoue K, Ukena K, Tsutsui K, Silver R (2006) Identification and characterization of a gonadotropin-inhibitory system in the brains of mammals. Proc Nat Acad Sci USA 103:2410–2415

    Article  PubMed  CAS  Google Scholar 

  • Kriegsfeld LJ, Gibson EM, Williams WP III, Zhao S, Mason AO, Bentley GE, Tsutsui K (2010) The roles of RFamide-related peptide-3 in mammalian reproductive function and behaviour. J Neuroendocrinol 22:692–700

    Article  PubMed  CAS  Google Scholar 

  • Leung PKC, Cheng CK (2004) GnRH as an autocrine regulator in the human ovary. In: Leung PKC, Adashi EY (eds) The ovary. Elsevier Academic press, Amsterdam, pp 289–304

    Google Scholar 

  • Leung PC, Cheng CK, Zhu XM (2003) Multi-factorial role of GnRH-I and GnRH-II in the human ovary. Mol Cell Endocrinol 202:145–153

    Article  PubMed  CAS  Google Scholar 

  • Li WI, Jiao S, Chin PP (1993) Immunoreactive gonadotropin-releasing hormone in porcine reproductive tissues. Peptides 14:543–549

    Article  PubMed  CAS  Google Scholar 

  • Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the folin phenol reagent. J Biol Chem 193:265–275

    PubMed  CAS  Google Scholar 

  • Metallinou C, Asimakopoulos B, Schroer A, Nikolettos N (2007) Gonadotropin-releasing hormone in the ovary. Reprod Sci 14:737

    Article  PubMed  CAS  Google Scholar 

  • Minaretzis D, Jakubowski M, Mortola JF, Pavlou SN (1995) Gonadotropin-releasing hormone receptor gene expression in human ovary and granulosa-lutein cells. J Clin Endocrinol Metab 80:430–434

    Article  PubMed  CAS  Google Scholar 

  • Osugi T, Ukena K, Bentley GE, O’Brien S, Moore IT, Wingfield JC, Tsutsui K (2004) Gonadotropin-inhibitory hormone in Gambel’s white-crowned sparrow (Zonotrichia leucophrys gambelii): cDNA identification, transcript localization and functional effects in laboratory and field experiments. J Endocrinol 182:33–42

    Article  PubMed  CAS  Google Scholar 

  • Parborell F, Dain L, Tesone M (2001) Gonadotropin-releasing hormone agonist affects rat ovarian follicle development by interfering with FSH and growth factors on the prevention of apoptosis. Mol Reprod Dev 60:241–247

    Article  PubMed  CAS  Google Scholar 

  • Parborell F, Pecci A, Gonzalez O, Vitale A, Tesone M (2002) Effects of a gonadotropin-releasing hormone agonist on rat ovarian follicle apoptosis: regulation by epidermal growth factor and the expression of Bcl-2-related genes. Biol Reprod 67:481–486

    Article  PubMed  CAS  Google Scholar 

  • Peng C, Fan NC, Ligier M, Vaananen J, Leung PC (1994) Expression and regulation of gonadotropin-releasing hormone (GnRH) and GnRH receptor messenger ribonucleic acids in human granulosa-luteal cells. Endocrinology 135:1740–1746

    Article  PubMed  CAS  Google Scholar 

  • Piquette GN, LaPolt PS, Oikawa M, Hsueh AJ (1991) Regulation of luteinizing hormone receptor messenger ribonucleic acid levels by gonadotropins, growth factors, and gonadotropin-releasing hormone in cultured rat granulosa cells. Endocrinology 128:2449–2456

    Article  PubMed  CAS  Google Scholar 

  • Richards JS (1994) Hormonal control of gene expression in the ovary. Endocr Rev 15:725–751

    PubMed  CAS  Google Scholar 

  • Schirman-Hildesheim TD, Bar T, Ben-Aroya N, Koch Y (2005) Differential gonadotropin-releasing hormone (GnRH) and GnRH receptor messenger ribonucleic acid expression patterns in different tissues of the female rat across the estrous cycle. Endocrinology 146:3401–3408

    Article  PubMed  CAS  Google Scholar 

  • Schirman-Hildesheim TD, Ben-Aroya N, Koch Y (2006) Daily GnRH and GnRH-receptor mRNA expression in the ovariectomized and intact rat. Mol Cell Endocrinol 252:120–125

    Article  PubMed  CAS  Google Scholar 

  • Sengupta A, Chakrabarti N, Sridaran R (2008) Presence of immunoreactive gonadotropin releasing hormone (GnRH) and its receptor (GnRHR) in rat ovary during pregnancy. Mol Reprod Dev 75:1031–1044

    Article  PubMed  CAS  Google Scholar 

  • Sethi S, Tsutsui K, Chaturvedi CM (2010a) Temporal phase relation of circadian neural oscillations alters RFamide-related peptide-3 and testicular function in the mouse. Neuroendocrinology 91:189–199

    Article  PubMed  CAS  Google Scholar 

  • Sethi S, Tsutsui K, Chaturvedi CM (2010b) Age-dependent variation in the RFRP-3 neurons is inversely correlated with gonadal activity of mice. Gen Comp Endocrinol 168:326–332

    Article  PubMed  CAS  Google Scholar 

  • Singh P, Krishna A, Sridaran R (2007) Localization of gonadotrophin-releasing hormone I, bradykinin and their receptors in the ovaries of non-mammalian vertebrates. Reproduction 133:969–981

    Article  PubMed  CAS  Google Scholar 

  • Singh P, Krishna A, Sridaran R, Tsutsui K (2008) Changes in GnRH I, bradykinin and their receptors and GnIH in the ovary of Calotes versicolor during reproductive cycle. Gen Comp Endocrinol 159:158–169

    Article  PubMed  CAS  Google Scholar 

  • Singh P, Krishna A, Tsutsui K (2011) Effects of Gonadotropin-inhibitory hormone on folliculogenesis and steroidogenesis of cyclic mice. Fert Stert 95:1397–1404

    Article  CAS  Google Scholar 

  • Sridaran R, Hisheh S, Dharmarajan AM (1998) Induction of apoptosis by a gonadotropin-releasing hormone agonist during early pregnancy in the rat. Apoptosis 3:51–57

    Article  PubMed  CAS  Google Scholar 

  • Sridaran R, Philip GH, Li H, Culty M, Liu Z, Stocco DM, Papadopoulos V (1999) GnRH agonist treatment decreases progesterone synthesis, luteal peripheral benzodiazepine receptor mRNA, ligand binding and steroidogenic acute regulatory protein expression during pregnancy. J Mol Endocrinol 22:45–54

    Article  PubMed  CAS  Google Scholar 

  • Srivastava RK, Krishna A, Sridaran R (1995) Follicular development, steroidogenesis and gonadotrophin secretion in response to long-term treatment with a gonadotrophin-releasing hormone agonist in the rat. J Endocrinol 146:349–357

    Article  PubMed  CAS  Google Scholar 

  • Tilly JL, Billig H, Kowalski KI, Hsueh AJ (1992) Epidermal growth factor and basic fibroblast growth factor suppress the spontaneous onset of apoptosis in cultured rat ovarian granulosa cells and follicles by a tyrosine kinase-dependent mechanism. Mol Endocrinol 6:1942–1950

    Article  PubMed  CAS  Google Scholar 

  • Tsutsui K (2009) A new key neurohormone controlling reproduction, gonadotropin-inhibitory hormone (GnIH): biosynthesis, mode of action and functional significance. Progr Neurobiol 88:76–88

    Article  CAS  Google Scholar 

  • Tsutsui K, Bentley GE (2008) Gonadotropin-inhibitory hormone (GnIH): biosynthesis, mode of action and functional significance in birds. Avian Biol Res 1:177–188

    Google Scholar 

  • Tsutsui K, Saigoh E, Ukena K, Teranishi H, Fujisawa Y, Kikuchi M, Ishii S, Sharp PJ (2000) A novel avian hypothalamic peptide inhibiting gonadotropin release. Biochem Biophys Res Commun 275:661–667

    Article  PubMed  CAS  Google Scholar 

  • Tsutsui K, Saigoh E, Yin H, Ubuka T, Chowdhury VS, Osugi T, Ukena K, Sharp PJ, Wingfield JC, Bentley GE (2009) A new key neurohormone controlling reproduction, gonadotrophin-inhibitory hormone in birds: discovery, progress and prospects. J Neuroendocrinol 21:271–275

    Article  PubMed  CAS  Google Scholar 

  • Tsutsui K, Bentley GE, Bedecarrats G, Osugi T, Ubuka T, Kriegsfeld LJ (2010a) Gonadotropin-inhibitory hormone (GnIH) and its control of central and peripheral reproductive function. Frontiers Neuroendocrinol 31:284–295

    Article  CAS  Google Scholar 

  • Tsutsui K, Bentley GE, Kriegsfeld LJ, Osugi T, Seong JY, Vaudry H (2010b) Discovery and evolutionary history of gonadotrophin-inhibitory hormone and kisspeptin: new key neuropeptides controlling reproduction. J Neuroendocrinol 22:716–727

    PubMed  CAS  Google Scholar 

  • Ubuka T, Kim S, Huang Yc, Reid J, Jiang J, Osugi T, Chowdhury VS, Tsutsui K, Bentley GE (2008) Gonadotropin-inhibitory hormone neurons interact directly with gonadotropin-releasing hormone-I and -II neurons in European starling brain. Endocrinology 149:268–278

    Article  PubMed  CAS  Google Scholar 

  • Ubuka T, Morgan K, Pawson AJ, Osugi T, Chowdhury VS, Minakata H, Tsutsui K, Millar RP, Bentley GE (2009) Identification of human GnIH homologs, RFRP-1 and RFRP-3, and the cognate receptor, GPR147 in the human hypothalamic pituitary axis. PLoS ONE 4:1–7

    Article  Google Scholar 

  • Ukena K, Tsutsui K (2001) Distribution of novel RFamide-related peptide-like immunoreactivity in the mouse central nervous system. Neurosci Lett 300:153–156

    Article  PubMed  CAS  Google Scholar 

  • Ukena K, Iwakoshi E, Minakata H, Tsutsui K (2002) A novel rat hypothalamic RFamide-related peptide identified by immunoaffinity chromatography and mass spectrometry. FEBS Lett 512:255–258

    Article  PubMed  CAS  Google Scholar 

  • Vaananen JE, Tong BL, Vaananen CM, Chan IH, Yuen BH, Leung PC (1997) Interaction of prostaglandin F2alpha and gonadotropin-releasing hormone on progesterone and estradiol production in human granulosa-luteal cells. Biol Reprod 57:1346–1353

    Article  PubMed  CAS  Google Scholar 

  • Whitelaw PF, Eidne KA, Sellar R, Smyth CD, Hillier SG (1995) Gonadotropin-releasing hormone receptor messenger ribonucleic acid expression in rat ovary. Endocrinology 136:172–179

    Article  PubMed  CAS  Google Scholar 

  • Zhao S, Zhu E, Yang C, Bentley GE, Tsutsui K, Kriegsfeld LJ (2010) RFamide-related peptide and messenger ribonucleic acid expression in mammalian testis: association with the spermatogenic cycle. Endocrinology 151:617–627

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported in part by Grant-in aid from UGC, New Delhi to (F. 34-420/2008 SR) AK; Scientific research from the Ministry of Education, Science and Culture, Japan (15207007, 16086206 and 18107002) to KT and grants from NIH, USA (HD41749, HD52155 and RR03034) to RS. PS is thankful to CSIR, India for providing research fellowships.

Conflict of Interest

The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Amitabh Krishna.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Singh, P., Krishna, A., Sridaran, R. et al. Immunohistochemical localization of GnRH and RFamide-related peptide-3 in the ovaries of mice during the estrous cycle. J Mol Hist 42, 371–381 (2011). https://doi.org/10.1007/s10735-011-9340-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10735-011-9340-8

Keywords

Navigation