Stress rapidly suppresses in vivo LH pulses and increases activation of RFRP-3 neurons in male mice

in Journal of Endocrinology
Correspondence should be addressed to A S Kauffman:
Restricted access

48-hour access to this article

USD $30.00

Restraint stress is a psychosocial stressor that suppresses reproductive status, including LH pulsatile secretion, but the neuroendocrine mechanisms underlying this inhibition remains unclear. Reproductive neural populations upstream of gonadotropin-releasing hormone (GnRH) neurons, such as kisspeptin, neurokinin B and RFRP-3 (GnIH) neurons, are possible targets for psychosocial stress to inhibit LH pulses, but this has not been well examined, especially in mice in which prior technical limitations prevented assessment of in vivo LH pulse secretion dynamics. Here, we examined whether one-time acute restraint stress alters in vivo LH pulsatility and reproductive neural populations in male mice, and what the time-course is for such alterations. We found that endogenous LH pulses in castrated male mice are robustly and rapidly suppressed by one-time, acute restraint stress, with suppression observed as quickly as 12–18 min. This rapid LH suppression parallels with increased in vivo corticosterone levels within 15 min of restraint stress. Although Kiss1, Tac2 and Rfrp gene expression in the hypothalamus did not significantly change after 90 or 180 min restraint stress, arcuate Kiss1 neural activation was significantly decreased after 180 min. Interestingly, hypothalamic Rfrp neuronal activation was strongly increased at early times after restraint stress initiation, but was attenuated to levels lower than controls by 180 min of restraint stress. Thus, the male neuroendocrine reproductive axis is quite sensitive to short-term stress exposure, with significantly decreased pulsatile LH secretion and increased hypothalamic Rfrp neuronal activation occurring rapidly, within minutes, and decreased Kiss1 neuronal activation also occurring after longer stress durations.


An official journal of

Society for Endocrinology



  • View in gallery

    Restraint stress inhibits pulsatile LH in gonadectomized (GDX) male mice. (A, B and C) represent three individual control mice and (D, E and F) represent three individual stress mice. Each graph represents an individual male mouse with blood collected every 6 min for 180 min. The initial 90 min period (−90 to 0 min) represents the pre-stress (baseline) period and the gray boxes (time 0 to 90 min) represent the post (stress) period. Pulse peaks are indicated by *.

  • View in gallery

    LH pulse parameters in GDX no stress (control) and stress male mice. The following LH parameters were analyzed: (A) pulse frequency, shown as pulses per 90 min, (B) interpulse interval, (C) pulse amplitude, (D) pulse peak levels and (E) basal LH levels. White bars are the pre-period (baseline) of blood collection (−90 to 0 min) and the black bars are the post period (stress) of blood collection (0 to 90 min). *P < 0.05 vs baseline (pre-stress) value.

  • View in gallery

    (A) Mean LH measured pre-stress (baseline; white bars) and post-stress initiation (black bars) in no stress (control) and stressed male mice. (B) Detailed time-course of LH changes after stress exposure, measured at 6 min intervals to determine how soon LH decreased following initiation of restraint stress. Mean blood LH levels before (−12 to 0) and after (0 to 24) restraint stress. White bars are control mice and black bars are restraint stressed mice. *P < 0.05.

  • View in gallery

    CORT secretion time-course in non-stressed control (closed, black circles) and acutely restrained male mice (open, white circles). Time 0 min represents time of restraint stress initiation in the restraint mice. *P < 0.05 control vs restraint mice, within time point.

  • View in gallery

    Acute restraint stress has no effect on arcuate Kiss1 or Tac2 cell number in male mice. (A) Representative image of single-label ISH for arcuate Kiss1 in a no stress control mouse. (B) Mean (± s.e.m.) Kiss1 cell number in no stress control (white bar) and stressed groups (90 and 180 min; black bars). (C) Representative images of single-label ISH for Tac2 in a no stress control mouse. (D) Mean (± s.e.m.) Tac2 cell number in no stress control (white bar) and stressed groups (90 and 180 min; black bars). *P < 0.05 vs no stress controls. 3V, third ventricle; ARC, arcuate. Scale bar = 100 µm.

  • View in gallery

    Restraint stress does not change Rfrp coexpression in male mice. (A) Representative image of single-label ISH for Rfrp in the DMN of a no stress control male. (B) Mean (± s.e.m.) Rfrp cell number in no stress control (white bar) and stressed groups (90 and 180 min; black bars). *P < 0.05 vs no stress controls. 3V, third ventricle; DMN, dorsal-medial nucleus. Scale bar = 100 µm.

  • View in gallery

    Arcuate Kiss1 neural activation in stress vs no stress control male mice. (A) Representative images of Kiss1 (red fluorescence) and cfos mRNA (silver grains) gene expression in a no stress control male. Yellow arrowheads represent coexpression of Kiss1 + cfos; blue arrowheads denote Kiss1 neurons not coexpressing cfos mRNA. (B) Quantification of coexpression of Kiss1 and cfos mRNA in the arcuate of male mice. *P < 0.05, compared to no stress controls. Scale bar = 25 µm.

  • View in gallery

    DMN Rfrp neural activation in stress vs no stress control male mice. (A) Representative images of Rfrp (red fluorescence) and cfos mRNA (silver grains) gene expression in a no stress control male. Yellow arrowheads represent coexpression of Rfrp + cfos mRNA; blue arrowheads denote Rfrp neurons not coexpressing cfos mRNA. (B) Quantification of coexpression of Rfrp and cfos mRNA in the DMN of male mice. *P < 0.05, compared to no stress controls. Scale bar = 50 µm.


AndersonGMRelfHLRizwanMZEvansJJ 2009 Central and peripheral effects of RFamide-related peptide-3 on luteinizing hormone and prolactin secretion in rats. Endocrinology 150 18341840. (

ChenMDO’ByrneKTChiappiniSEHotchkissJKnobilE 1992 Hypoglycemic ‘stress’ and gonadotropin-releasing hormone pulse generator activity in the rhesus monkey: role of the ovary. Neuroendocrinology 56 666673. (

ChenMDOrdogTO’ByrneKTGoldsmithJRConnaughtonMAKnobilE 1996 The insulin hypoglycemia-induced inhibition of gonadotropin-releasing hormone pulse generator activity in the rhesus monkey: roles of vasopressin and corticotropin-releasing factor. Endocrinology 137 20122021. (

ClarkeIJSariIPQiYSmithJTParkingtonHCUbukaTIqbalJLiQTilbrookAMorganKet al. 2008 Potent action of RFamide-related peptide-3 on pituitary gonadotropes indicative of a hypophysiotropic role in the negative regulation of gonadotropin secretion. Endocrinology 149 58115821. (

ClarksonJHanSYPietRMcLennanTKaneGMNgJPorteousRWKimJSColledgeWHIremongerKJet al. 2017 Definition of the hypothalamic GnRH pulse generator in mice. PNAS 114 E10216E10223. (

DemuraRSuzukiTNakamuraSKomatsuHOdagiriEDemuraH 1989 Effect of immobilization stress on testosterone and inhibin in male rats. Journal of Andrology 10 210213. (

DucretEGaidamakaGHerbisonAE 2010 Electrical and morphological characteristics of anteroventral periventricular nucleus kisspeptin and other neurons in the female mouse. Endocrinology 151 22232232. (

FensterLKatzDFWyrobekAJPieperCRempelDMOmanDSwanSH 1997 Effects of psychological stress on human semen quality. Journal of Andrology 18 194202. (

GeorgeJTHendrikseMVeldhuisJDClarkeIJAndersonRAMillarRP 2017 Effect of gonadotropin-inhibitory hormone on luteinizing hormone secretion in humans. Clinical Endocrinology 86 731738. (

GoodmanRLHilemanSMNestorCCPorterKLConnorsJMHardySLMillarRPCerneaMCoolenLMLehmanMN 2013 Kisspeptin, neurokinin B, and dynorphin act in the arcuate nucleus to control activity of the GnRH pulse generator in ewes. Endocrinology 154 42594269. (

GronowskiAMRotweinP 1995 Rapid changes in gene expression after in vivo growth hormone treatment. Endocrinology 136 47414748. (

HaasCAReddingtonMKreutzbergGW 1991 Calcitonin gene-related peptide stimulates the induction of c-fos gene expression in rat astrocyte cultures. European Journal of Neuroscience 3 708712. (

HamamuraMNunezDJLengGEmsonPCKiyamaH 1992 c-fos may code for a common transcription factor within the hypothalamic neural circuits involved in osmoregulation. Brain Research 572 4251. (

Handa RJBurgess LHKerr JEO’Keefe JA 1994 Gonadal steroid hormone receptors and sex differences in the hypothalamo-pituitary-adrenal axis. Hormones and Behavior 28 464476. (

ImakiTShibasakiTHottaMDemuraH 1992 Early induction of c-fos precedes increased expression of corticotropin-releasing factor messenger ribonucleic acid in the paraventricular nucleus after immobilization stress. Endocrinology 131 240246. (

IwasaTMatsuzakiTTungalagsuvdAMunkhzayaMKawamiTNikiHKatoTKuwaharaAUemuraHYasuiTet al. 2014 Hypothalamic Kiss1 and RFRP gene expressions are changed by a high dose of lipopolysaccharide in female rats. Hormones and Behavior 66 309316. (

JohnsonMATsutsuiKFraleyGS 2007 Rat RFamide-related peptide-3 stimulates GH secretion, inhibits LH secretion, and has variable effects on sex behavior in the adult male rat. Hormones and Behavior 51 171180. (

Kinsey-JonesJSLiXFKnoxAMWilkinsonESZhuXLChaudharyAAMilliganSRLightmanSLO’ByrneKT 2009 Down-regulation of hypothalamic kisspeptin and its receptor, Kiss1r, mRNA expression is associated with stress-induced suppression of luteinising hormone secretion in the female rat. Journal of Neuroendocrinology 21 2029. (

KirbyEDGeraghtyACUbukaTBentleyGEKauferD 2009 Stress increases putative gonadotropin inhibitory hormone and decreases luteinizing hormone in male rats. PNAS 106 1132411329. (

KriegsfeldLJMeiDFBentleyGEUbukaTMasonAOInoueKUkenaKTsutsuiKSilverR 2006 Identification and characterization of a gonadotropin-inhibitory system in the brains of mammals. PNAS 103 24102415. (

LehmanMNCoolenLMGoodmanRL 2010 Minireview: kisspeptin/neurokinin B/dynorphin (KNDy) cells of the arcuate nucleus: a central node in the control of gonadotropin-releasing hormone secretion. Endocrinology 151 34793489. (

LiXFEdwardJMitchellJCShaoBBowesJECoenCWLightmanSLO’ByrneKT 2004 Differential effects of repeated restraint stress on pulsatile lutenizing hormone secretion in female Fischer, Lewis and Wistar rats. Journal of Neuroendocrinology 16 620627. (

LiXFBoweJEKinsey-JonesJSBrainSDLightmanSLO’ByrneKT 2006 Differential role of corticotrophin-releasing factor receptor types 1 and 2 in stress-induced suppression of pulsatile luteinising hormone secretion in the female rat. Journal of Neuroendocrinology 18 602610. (

Lopez-CalderonAAriznavarretaCGonzalez-QuijanoMITresguerresJACalderonMD 1991 Stress induced changes in testis function. Journal of Steroid Biochemistry and Molecular Biology 40 473479. (

MannDROrrTE 1990 Effect of restraint stress on gonadal proopiomelanocortin peptides and the pituitary-testicular axis in rats. Life Sciences 46 16011609. (

OrrTEMannDR 1990 Effects of restraint stress on plasma LH and testosterone concentrations, Leydig cell LH/hCG receptors, and in vitro testicular steroidogenesis in adult rats. Hormones and Behavior 24 324341. (

OsugiTUkenaKBentleyGEO’BrienSMooreITWingfieldJCTsutsuiK 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. Journal of Endocrinology 182 3342. (

PolingMCKimJDhamijaSKauffmanAS 2012 Development, sex steroid regulation, and phenotypic characterization of RFamide-related peptide (Rfrp) gene expression and RFamide receptors in the mouse hypothalamus. Endocrinology 153 18271840. (

PolingMCLuoEYKauffmanAS 2017 Sex differences in steroid receptor coexpression and circadian-timed activation of kisspeptin and RFRP-3 neurons may contribute to the sexually dimorphic basis of the LH surge. Endocrinology 158 35653578. (

RivierCRivestS 1991 Effect of stress on the activity of the hypothalamic-pituitary-gonadal axis: peripheral and central mechanisms. Biology of Reproduction 45 523532. (

SemaanSJKauffmanAS 2015 Daily successive changes in reproductive gene expression and neuronal activation in the brains of pubertal female mice. Molecular and Cellular Endocrinology 401 8497. (

StephensSBChahalNMunaganuruNParraRAKauffmanAS 2016 Estrogen stimulation of kiss1 expression in the medial amygdala involves estrogen receptor-alpha but not estrogen receptor-beta. Endocrinology 157 40214031. (

StephensSBZRouseMLTolsonKPLiawRBParraRAChahalNKauffmanAS 2017 Effects of selective deletion of tyrosine hydroxylase from kisspeptin cells on puberty and reproduction in male and female mice. eNeuro 4 112.

TakumiKIijimaNHigoSOzawaH 2012 Immunohistochemical analysis of the colocalization of corticotropin-releasing hormone receptor and glucocorticoid receptor in kisspeptin neurons in the hypothalamus of female rats. Neuroscience Letters 531 4045. (

TilbrookAJCannyBJSerapigliaMDAmbroseTJClarkeIJ 1999 Suppression of the secretion of luteinizing hormone due to isolation/restraint stress in gonadectomised rams and ewes is influenced by sex steroids. Journal of Endocrinology 160 469481. (

TilbrookAJTurnerAIClarkeIJ 2000 Effects of stress on reproduction in non-rodent mammals: the role of glucocorticoids and sex differences. Reproductive Biology 5 105113.

TilbrookAJTurnerAIClarkeIJ 2002 Stress and reproduction: central mechanisms and sex differences in non-rodent species. Stress 5 83100. (

ViauVMeaneyMJ 1991 Variations in the hypothalamic-pituitary-adrenal response to stress during the estrous cycle in the rat. Endocrinology 129 25032511. (

WagenmakerERBreenKMOakleyAETilbrookAJKarschFJ 2009 Psychosocial stress inhibits amplitude of gonadotropin-releasing hormone pulses independent of cortisol action on the type II glucocorticoid receptor. Endocrinology 150 762769. (

WeaverDRReppertSM 1995 Definition of the developmental transition from dopaminergic to photic regulation of c-fos gene expression in the rat suprachiasmatic nucleus. Brain Research. Molecular Brain Research 33 136148. (

YangJASongCIHughesJKKreismanMJParraRAHaisenlederDJKauffmanASBreenKM 2017 Acute psychosocial stress inhibits LH pulsatility and kiss1 neuronal activation in female mice. Endocrinology 158 37163723. (

Index Card


Google Scholar

Related Articles



All Time Past Year Past 30 Days
Abstract Views 99 99 65
Full Text Views 282 282 33
PDF Downloads 90 90 5