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1.  Prenatal PCBs Disrupt Early Neuroendocrine Development of the Rat Hypothalamus 
Neonatal exposure to endocrine disrupting chemicals (EDCs) such as polychlorinated biphenyls (PCBs) can interfere with hormone-sensitive developmental processes, including brain sexual differentiation. We hypothesized that disruption of these processes by gestational PCB exposure would be detectable as early as the day after birth (postnatal day (P) 1) through alterations in hypothalamic gene and protein expression. Pregnant Sprague-Dawley rats were injected twice, once each on gestational days 16 and 18, with one of the following: DMSO vehicle; the industrial PCB mixture Aroclor 1221 (A1221); a reconstituted mixture of the three most prevalent congeners found in humans: PCB138, PCB153 and PCB180; or estradiol benzoate (EB). On P1, litter composition, anogenital distance (AGD) and body weight were assessed. Pups were euthanized for immunohistochemistry of estrogen receptor α (ERα) or TUNEL labeling of apoptotic cells, or quantitative PCR of 48 selected genes in the preoptic area (POA). We found that treatment with EB or A1221 had a sex-specific effect on developmental apoptosis in the neonatal anteroventral periventricular nucleus (AVPV), a sexually dimorphic hypothalamic region involved in the regulation of reproductive neuroendocrine function. In this region, exposed females had increased numbers of apoptotic nuclei, whereas there was no effect of treatment in males. For ERα, EB treatment increased immunoreactive cell numbers and density in the AVPV of both males and females, while A1221 and the PCB mixture had no effect. PCR analysis of gene expression in the POA identified nine genes that were significantly altered by prenatal EDC exposure, in a manner that varied by sex and treatment. These genes included brain-derived neurotrophic factor, GABAB receptors-1 and -2, IGF-1, kisspeptin receptor, NMDA receptor subunits NR2b and NR2c, prodynorphin, and TGFα. Collectively, these results suggest that the disrupted sexual differentiation of the POA by prenatal EDC exposures is already evident as early as the day after birth, effects that may change the trajectory of postnatal development and compromise adult reproductive function.
PMCID: PMC3060304  PMID: 21277884
Polychlorinated biphenyls (PCBs); endocrine disruption; hypothalamus; sexual differentiation; neuroendocrine development; estrogen receptor; developmental apoptosis; anteroventral periventricular nucleus; medial preoptic nucleus
2.  Age-related Changes in Hypothalamic Androgen Receptor and Estrogen Receptor α in Male Rats# 
The control of reproductive function involves actions of sex steroids upon their nuclear receptors in the hypothalamus and preoptic area (POA). Whether hypothalamic hormone receptors change their expression in aging male mammals has not been extensively pursued, although such changes may underlie functional losses in reproductive physiology occurring with aging. We performed a stereological analysis of immunoreactive androgen receptor (AR) and estrogen receptor alpha (ERα) cells in three POA nuclei of male Sprague-Dawley rats [anteroventral periventricular nucleus (AVPV), median preoptic area (MePO), and medial preoptic nucleus (MPN)], at young (3 mo), middle-aged (12 mo) and old (20 mo) ages. Serum testosterone and estradiol levels were assayed. Testosterone concentrations decreased significantly and progressively with aging. Estradiol concentrations were significantly higher in middle-aged than either young or old rats. Stereologic analyses of the POA demonstrated that AR-immunoreactive cell numbers and density in the AVPV, MePO and MPN were significantly higher in old compared with young or middle-aged rats. No change in the total number or density of ERα immunoreactive cells was detected with age, although when cells were subdivided by intensity of immunolabeling, the most heavily-labeled ERα cells increased in number with aging in the AVPV and MePO, and in density in the AVPV. There are several interpretations to our finding of substantially increased AR cell numbers during aging, including a potential compensatory up-regulation of the AR under diminished testosterone concentrations. These results provide further information about how the neural targets of steroid hormones change with advancing age.
PMCID: PMC2671934  PMID: 19051266
androgen receptor; estrogen receptor; aging; anteroventral periventricular nucleus (AVPV); median preoptic area (MePO); medial preoptic nucleus (MPN)
3.  Breeding status and social environment differentially affect the expression of sex steroid receptor and aromatase mRNA in the brain of female Damaraland mole-rats 
Frontiers in Zoology  2014;11:38.
The Damaraland mole-rat (Fukomys damarensis) is a eusocial, subterranean mammal, which exhibits an extreme reproductive skew with a single female (queen) monopolizing reproduction in each colony. Non-reproductive females in the presence of the queen are physiologically suppressed to the extent that they are anovulatory. This blockade is thought to be caused by a disruption in the normal gonadotropin-releasing hormone (GnRH) secretion from the hypothalamus. In order to understand the underlying physiological mechanisms of reproductive suppression in subordinate females we studied the expression of steroid hormone receptors and the androgen-converting enzyme aromatase in forebrain regions involved in the control of reproductive behaviour in female breeders and non-breeders from intact colonies. Additionally, we included in our analysis females that experienced the release from social suppression by being removed from the presence of the queen.
We found expression of androgen receptor, estrogen receptor α and aromatase in several forebrain regions of female Damaraland mole-rats. Their distribution matches previous findings in other mammals. Quantification of the hybridisation signal revealed that queens had increased expression of androgen receptors compared to non-breeders and removed non-breeders in most brain regions examined, which include the medial preoptic area (MPOA), the principal nucleus of the bed nucleus of the stria terminalis (BSTp), the ventromedial nucleus of the hypothalamus (VMH), the arcuate nucleus (ARC) and the medial amygdala (MeA). Furthermore, breeders had increased estrogen receptor α expression in the anteroventral periventricular nucleus (AVPV) and in the MeA, while aromatase expression in the AVPV was significantly reduced compared to non-breeders. Absence of social suppression was associated with increased androgen receptor expression in the ARC, increased estrogen receptor α expression in the MeA and BSTp and reduced aromatase expression in the AVPV.
This study shows that social suppression and breeding differentially affect the neuroendocrine phenotype of female Damaraland mole-rats. The differential expression pattern of estrogen receptor α and aromatase in the AVPV between breeders and non-breeders supports the view that this region plays an important role in mediating the physiological suppression in subordinate females.
PMCID: PMC4024188  PMID: 24839456
Androgen receptor; Estrogen receptor alpha; Aromatase; In situ hybridization; Fukomys damarensis; Social status
4.  An eGFP-expressing subpopulation of growth hormone secretagogue receptor cells are distinct from kisspeptin, tyrosine hydroxylase, and RFamide-related peptide neurons in mice 
Peptides  2013;47:45-53.
Ghrelin acts on the growth hormone secretagogue receptor (GHSR) in the brain to elicit changes in physiological functions. It is associated with the neural control of appetite and metabolism, however central ghrelin also affects fertility. Central ghrelin injection in rats suppresses luteinizing hormone (LH) concentrations and pulse frequency. Although ghrelin suppresses LH and regulates kisspeptin mRNA in the anteroventral periventricular/periventricular nucleus (AVPV/PeN), there is no neuroanatomical evidence linking GHSR neural circuits to kisspeptin neurons. In this study, we first determined coexpression of GHSR and GnRH neurons using a GHSR-eGFP reporter mouse line. Using dual-label immunohistochemistry, we saw no coexpression. GHSR-eGFP expressing cells were present in the AVPV/PeN and over 90% of these expressed estrogen receptor-α (ERα). Despite this, we observed no evidence of GHSR-eGFP/kisspeptin coexpressing neurons in the AVPV/PeN. To further examine the phenotype of GHSR-eGFP cells in the AVPV/PeN, we determined coexpression with tyrosine hydroxylase (TH) and showed virtually no coexpression in the AVPV/PeN (<2%). We also observed no coexpression of GHSR-eGFP and RFamide-related peptide-3 (RFRP3) neurons in the dorsomedial hypothalamic nucleus. Importantly, we observed that approximately half of the GHSR-eGFP cells in the AVPV coexpressed Ghsr mRNA (as determined by in situ hybridization) so these data should be interpreted accordingly. Although ghrelin influences the hypothalamic reproductive axis, our data using a GHSR-eGFP reporter suggests ghrelin regulates neurons expressing ERα but does not directly act on GnRH, kisspeptin, TH, or RFRP3 neurons, as little or no GHSR-eGFP coexpression was observed.
PMCID: PMC3762877  PMID: 23831041
ghrelin; GHSR; GFP; Kisspeptin; AgRP; reproduction
5.  Age-related LH surge dysfunction correlates with reduced responsiveness of hypothalamic anteroventral periventricular nucleus kisspeptin neurons to estradiol positive feedback in middle-aged rats 
Neuropharmacology  2009;58(1):314-320.
Female reproductive aging in rats is characterized by reduced gonadotropin releasing hormone (GnRH) neuronal activation under estradiol positive feedback conditions and a delayed and attenuated luteinizing hormone (LH) surge. The newly identified excitatory neuropeptide kisspeptin is proposed to be a critical mediator of the pubertal transition and the ovarian steroid-induced LH surge. We previously showed that estradiol induces less kisspeptin mRNA expression in the anterior hypothalamus [anatomical location of anteroventral periventricular nucleus (AVPV)] in middle-aged than in young rats and intrahypothalamic infusion of kisspeptin restores LH surge amplitude in middle-aged females. Thus, reduced kisspeptin neurotransmission may contribute to age-related LH surge abnormalities. This study tested the hypothesis that middle-aged females will also exhibit reduced numbers of kisspeptin immunopositive neurons in the AVPV under estradiol positive feedback conditions. Using immunohistochemistry, we demonstrate that middle-aged females primed with ovarian steroids have fewer AVPV kisspeptin immunopositive neurons than young females. Age did not affect kisspeptin mRNA expression in the pituitary, numbers of kisspeptin immunopositive neurons in the arcuate nucleus, or estradiol-dependent reductions in kisspeptin mRNA expression in the posterior hypothalamus (containing the arcuate nucleus). These data strongly suggest that age-related LH surge dysfunction results, in part, from a reduced sensitivity of AVPV kisspeptin neurons to estradiol and hence decreased availability of AVPV kisspeptin neurons to activate GnRH neurons under positive feedback conditions.
PMCID: PMC2901500  PMID: 19559035
Aging; Kisspeptin; GPR54; Hypothalamus; AVPV; Arcuate nucleus
6.  Short-term Alcohol Administration Alters KiSS-1 gene Expression in the Reproductive Hypothalamus of Prepubertal Female rats 
Kisspeptins bind to the G protein coupled receptor (GPR54), to activate hypothalamic luteinizing hormone releasing hormone (LHRH) secretion at the time of puberty. Alcohol (ALC) causes depressed prepubertal LHRH release, resulting in depressed luteinizing hormone (LH) secretion and delayed puberty. Because KiSS-1 and GPR54 are important to the onset of puberty, we assessed the effects of chronic ALC administration on basal expression of these puberty related genes within the reproductive hypothalamus, as well as hormones and transduction signaling pathways contributing to their activity.
Immature female rats were fed a liquid-diet containing ALC for 6 days beginning when 27 days old. Controls received either companion isocaloric liquid-diet or rat chow and water. Animals were decapitated on day 33, in the late juvenile stage of development. Blood was collected for assessment of serum hormone levels. Brain tissues containing the anteroventral periventricular (AVPV) and arcuate (ARC) nuclei were obtained for assessing expression of specific puberty related genes and proteins.
KiSS-1 mRNA levels in the AVPV and ARC nuclei were suppressed (p<0.01) in the ALC-treated rats. GPR54 gene and protein expressions were both modestly increased (p<0.05) in AVPV nucleus, but not in ARC nucleus. ALC exposure also resulted in suppressed serum levels of insulin-like growth factor −1 (IGF-1), LH and estradiol (E2). Since IGF-1, in the presence of E2, can induce expression of the KiSS-1 gene, we assessed the potential for ALC to alter IGF-1 signaling in the reproductive hypothalamus. IGF-1 receptor gene and protein expressions were not altered. However, protein expression of phosphorylated Akt, a transduction signal used by IGF-1, was suppressed in the AVPV (p<0.05) and ARC (p<0.01) nuclei.
ALC causes suppressed KiSS-1 gene expression in the reproductive hypothalamus; hence, contributing to this drug’s ability to cause suppressed LHRH secretion and disruption of the pubertal process. We suggest that this action, at least in part, is through altered IGF-1 signaling.
PMCID: PMC2950647  PMID: 19519717
Alcohol; KiSS-1 gene; Hypothalamus; Puberty
7.  Chronic Exposure to Anabolic Androgenic Steroids Alters Activity and Synaptic Function in Neuroendocrine Control Regions of the Female Mouse 
Neuropharmacology  2011;61(4):653-664.
Disruption of reproductive function is a hallmark of abuse of anabolic androgenic steroids (AAS) in female subjects. To understand the central actions of AAS, patch clamp recordings were made in estrous, diestrous and AAS-treated mice from gonadotropin releasing hormone (GnRH) neurons, neurons in the medial preoptic area (mPOA) and neurons in the anteroventroperiventricular nucleus (AVPV); regions known to provide GABAergic and kisspeptin inputs to the GnRH cells. Action potential (AP) frequency was significantly higher in GnRH neurons of estrous mice than in AAS-treated or diestrous animals. No significant differences in AAS-treated, estrous or diestrous mice were evident in the amplitude or kinetics of spontaneous postsynaptic currents (sPCSs), miniature PSCs or tonic currents mediated by GABAA receptors or in GABAA receptor subunit expression in GnRH neurons. In contrast, the frequency of GABAA receptor-mediated sPSCs in GnRH neurons showed an inverse correlation with AP frequency across the three hormonal states. Surprisingly, AP activity in the medial preoptic area (mPOA), a likely source of GABAergic afferents to GnRH cells, did not vary in concert with the sPSCs in the GnRH neurons. Furthermore, pharmacological blockade of GABAA receptors did not alter the pattern in which there was lower AP frequency in GnRH neurons of AAS-treated and diestrous versus estrous mice. These data suggest that AAS do not impose their effects either directly on GnRH neurons or on putative GABAergic afferents in the mPOA. AP activity recorded from neurons in kisspeptin-rich regions of the anteroventroperiventricular nucleus (AVPV) and the expression of kisspeptin mRNA and peptide did vary coordinately with AP activity in GnRH neurons. Our data demonstrate that AAS treatment imposes a “diestrous-like” pattern of activity in GnRH neurons and suggest that this effect may arise from suppression of presynaptic kisspeptin-mediated excitatory drive arising from the AVPV. The actions of AAS on neuroendocrine regulatory circuits may contribute the disruption of reproductive function observed in steroid abuse.
PMCID: PMC3130628  PMID: 21645530
Anabolic steroid; GnRH; GABAA receptor; drug abuse; kisspeptin; reproduction
8.  Effects of polychlorinated biphenyls on estrogen receptor-beta expression in the anteroventral periventricular nucleus. 
Environmental Health Perspectives  2003;111(10):1278-1282.
Polychlorinated biphenyls (PCBs) can disrupt the reproductive axis, particularly when the exposure occurs during the vulnerable developmental periods. Some effects of environmental endocrine disruptors such as PCBs may be exerted through binding to estrogen receptors (ERs). In this study we examined the endocrine-disrupting effects of Aroclor 1221 (a commercial PCB mixture), focusing on its actions on the ER-ss, which has been implicated in mediating effects of endocrine-disrupting chemicals. A low, ecologically relevant dose of Aroclor 1221 or vehicle (ethanol) was administered three times each to rat dams, on gestational day 16 and on postpartum days 1 and 4, a developmental period during which steroid hormones have permanent effects on adult brain structure and function. Effects on ER-ss cell number in the anteroventral periventricular nucleus (AVPV) were quantified; this sexually dimorphic nucleus of the brain is essential to female reproductive function. For comparison, we quantified ER-ss cell number in another hypothalamic region, the supraoptic nucleus (SON). Using a stereologic approach, we found that Aroclor 1221 caused a highly significant down-regulation of the number of ER-ss-expressing cells in the AVPV, but had no effect in the SON. Thus, PCB exposure has consequences for neural ER expression, and these findings have implications for wildlife and humans that have been exposed to environmental estrogens, particularly during the susceptible periods of early development.
PMCID: PMC1241606  PMID: 12896846
9.  Suppression of Kisspeptin Expression and Gonadotropic Axis Sensitivity Following Exposure to Inhibitory Day Lengths in Female Siberian Hamsters 
Hormones and behavior  2007;52(4):492-498.
To avoid breeding during unsuitable environmental or physiological circumstances, the reproductive axis adjusts its output in response to fluctuating internal and external conditions. The ability of the reproductive system to alter its activity appropriately in response to these cues has been well established. However, the means by which reproductively relevant cues are interpreted, integrated, and relayed to the reproductive axis remain less well specified. The neuropeptide kisspeptin has been shown to be a potent positive stimulator of the hypothalamo-pituitary-gonadal (HPG) axis, suggesting a possible neural locus for the interpretation/integration of these cues. Because a failure to inhibit reproduction during winter would be maladaptive for short-lived female rodents, female Siberian hamsters (Phodopus sungorus) housed in long and short days hamsters were examined. In long, ‘summer’ photoperiods, kisspeptin is highly expressed in the anteroventral periventricular nucleus (AVPV), with low expression in the arcuate nucleus (Arc). A striking reversal in this pattern is observed in animals held in short, ‘winter’ photoperiods, with negligible kisspeptin expression in the AVPV and marked staining in the Arc. Although all studies to date suggest that both populations act to stimulate the reproductive axis, these contrasting expression patterns of AVPV and Arc kisspeptin suggest disparate roles for these two cell populations. Additionally, we found that the stimulatory actions of exogenous kisspeptin are blocked by acyline, a gonadotropin-releasing hormone (GnRH) receptor antagonist, suggesting an action of kisspeptin on the GnRH system rather than pituitary gonadotropes. Finally, females held in short day lengths exhibit a reduced response to exogenous kisspeptin treatment relative to long-day animals. Together, these findings indicate a role for kisspeptin in the AVPV and Arc as an upstream integration center for reproductively-relevant stimuli and point to a dual mechanism of reproductive inhibition in which kisspeptin expression is reduced concomitant with reduced sensitivity of the HPG axis to this peptide.
PMCID: PMC2717891  PMID: 17706968
metastin; GPR54; photoperiod; Siberian hamster; seasonal; reproduction
10.  AVPV neurons containing estrogen receptor-beta in adult male rats are influenced by soy isoflavones 
BMC Neuroscience  2007;8:13.
Isoflavones, the most abundant phytoestrogens in soy foods, are structurally similar to 17beta-estradiol. It is known that 17beta-estradiol induces apoptosis in anteroventral periventricular nucleus (AVPV) in rat brain. Also, there is evidence that consumption of soy isoflavones reduces the volume of AVPV in male rats. Therefore, in this study, we examined the influence of dietary soy isoflavones on apoptosis in AVPV of 150 day-old male rats fed either a soy isoflavone-free diet (Phyto-free) or a soy isoflavone-rich diet (Phyto-600).
The occurrence of apoptosis in AVPV was examined by TUNEL staining. The incidence of apoptosis was about 10 times higher in the Phyto-600 group (33.1 ± 1.7%) than in the Phyto-free group (3.6 ± 1.0%). Furthermore, these apoptotic cells were identified as neurons by dual immunofluorescent staining of GFAP and NeuN as markers of astrocytes and neurons, respectively. Then the dopaminergic neurons in AVPV were detected by immunohistochemistry staining of tyrosine hydroxylase (TH). No significant difference in the number of TH neurons was observed between the diet treatment groups. When estrogen receptor (ER) alpha and beta were examined by immunohistochemistry, we observed a 22% reduction of ERbeta-positive cell numbers in AVPV with consumption of soy isoflavones, whereas no significant change in ERalpha-positive cell numbers was detected. Furthermore, almost all the apoptotic cells were ERbeta-immunoreactive (ir), but not ERalpha-ir. Last, subcutaneous injections of equol (a major isoflavone metabolite) that accounts for approximately 70–90% of the total circulating plasma isoflavone levels did not alter the volume of AVPV in adult male rats.
In summary, these findings provide direct evidence that consumption of soy isoflavones, but not the exposure to equol, influences the loss of ERbeta-containing neurons in male AVPV.
PMCID: PMC1797051  PMID: 17266774
11.  Fibroblast Growth Factor Signaling Deficiencies Impact Female Reproduction and Kisspeptin Neurons in Mice1 
Biology of Reproduction  2012;86(4):119.
Fibroblast growth factor (FGF) signaling is essential for the development of the gonadotropin-releasing hormone (GnRH) system. Mice harboring deficiencies in Fgf8 or Fgf receptor 1 (Fgfr1) suffer a significant loss of GnRH neurons, but their reproductive phenotypes have not been examined. This study examined if female mice hypomorphic for Fgf8, Fgfr1, or both (compound hypomorphs) exhibited altered parameters of pubertal onset, estrous cyclicity, and fertility. Further, we examined the number of kisspeptin (KP)-immunoreactive (ir) neurons in the anteroventral periventricular/periventricular nuclei (AVPV/PeV) of these mice to assess if changes in the KP system, which stimulates the GnRH system, could contribute to the reproductive phenotypes. Single hypomorphs (Fgfr1+/− or Fgf8+/−) had normal timing for vaginal opening (VO) but delayed first estrus. However, after achieving the first estrus, they underwent normal expression of estrous cycles. In contrast, the compound hypomorphs underwent early VO and normal first estrus, but had disorganized estrous cycles that subsequently reduced their fertility. KP immunohistochemistry on Postnatal Day 15, 30, and 60 transgenic female mice revealed that female compound hypomorphs had significantly more KP-ir neurons in the AVPV/PeV compared to their wild-type littermates, suggesting increased KP-ir neurons may drive early VO but could not maintain the cyclic changes in GnRH neuronal activity required for female fertility. Overall, these data suggest that Fgf signaling deficiencies differentially alter the parameters of female pubertal onset and cyclicity. Further, these deficiencies led to changes in the AVPV/PeV KP-ir neurons that may have contributed to the accelerated VO in the compound hypomorphs.
Single or combined deficiencies in Fgfr1 and Fgf8 differentially alter reproduction and the kisspeptin system in female mice.
PMCID: PMC3338661  PMID: 22278983
female infertility; gonadal function; gonadotropin-releasing hormone (GnRH/GnRH receptor); kisspeptin
12.  Sexual Experience Changes Sex Hormones But Not Hypothalamic Steroid Hormone Receptor Expression in Young and Middle-aged Male Rats 
Hormones and behavior  2009;56(3):299-308.
Testosterone is well known to regulate sexual behavior in males, but this is dependent upon prior sexual experience. Aging is associated with decreased libido and changes in testosterone, but the role of experience in these age-related processes has not been systematically studied. We examined effects of age and sexual experience on serum hormones (total testosterone, free testosterone, estradiol, LH) and on numbers of androgen receptor (AR) and estrogen receptor α (ERα) immunoreactive cells in the hypothalamus. Extensive sexual experience was given to male rats at 4 months of age. Rats were euthanized at either 4 months (young) or 12 months (middle-aged (MA)). Comparable sexually naïve male rats were handled and placed into the testing arena but did not receive any sexual experience. Thus, we had four groups: young-naïve, young-experienced, MA-naïve and MA-experienced. Serum hormone levels were assayed, and numbers of AR and ERα cells were quantified stereologically in the medial preoptic nucleus (MPN) and the anteroventral periventricular nucleus (AVPV). Sexually experienced males had significantly elevated serum testosterone and free testosterone in both age groups. Both total and free testosterone were higher, and estradiol lower, in middle-aged than young rats. Experience did not alter either AR or ERα expression in the preoptic brain regions studied. Aging was associated with increased expression of AR, but no change in ERα. These results show that sexual experience can induce short-term and long-term alterations in serum hormones but these effects are not manifested upon their receptors in the hypothalamus.
PMCID: PMC2739266  PMID: 19559704
androgen receptor; estrogen receptor α; sexual experience; aging; anteroventral periventricular nucleus (AVPV); medial preoptic nucleus (MPN)
13.  Shift in Kiss1 cell activity requires estrogen receptor α 
Reproductive function requires timely secretion of gonadotropin releasing hormone, which is controlled by a complex excitatory/inhibitory network influenced by sex steroids. Kiss1 neurons are fundamental players in this network, but it is currently unclear whether different conditions of circulating sex steroids directly alters Kiss1 neuronal activity. Here, we show that Kiss1 neurons in the anteroventral periventricular and anterior periventricular nuclei (AVPV/PeN) of males and females exhibit a bimodal resting membrane potential (RMP) influenced by KATP channels, suggesting the presence of two neuronal populations defined as Type I (irregular firing patterns) and Type II (quiescent). Kiss1 neurons in the arcuate nucleus (Arc) are also composed of firing and quiescent cells, but unlike AVPV/PeN neurons, the range of RMPs did not follow a bimodal distribution. Moreover, Kiss1 neuronal activity in the AVPV/PeN, but not in the Arc, is sexually dimorphic. In females, estradiol shifts the firing pattern of AVPV/PeN Kiss1 neurons and alters cell capacitance and spontaneous inhibitory postsynaptic potentials (IPSCs) amplitude of AVPV/PeN and Arc Kiss1 populations in an opposite manner. Notably, mice with selective deletion of estrogen receptor α (ERα) from Kiss1 neurons show cellular activity similar to that observed in ovariectomized females, suggesting that estradiol-induced changes in Kiss1 cellular properties require ERα. We also show that female prepubertal Kiss1 neurons are under higher inhibitory influence while all AVPV/PeN Kiss1 neurons are spontaneously active. Collectively, our findings indicate that changes in cellular activity may underlie Kiss1 action in pubertal initiation and female reproduction.
PMCID: PMC3713640  PMID: 23407940
HPG axis; puberty; hypothalamus; neuroendocrine regulation; estrogen feedback
14.  Neonatal Exposure to 17α-Ethinyl Estradiol Affects Kisspeptin Expression and LH-Surge Level in Female Rats 
Contamination of estrogenic compounds disrupts endocrinological and neurological reproductive systems in animals. Neonatal exposure to 17α-ethinyl estradiol (EE) induced an abnormal estrous cycle at postnatal day (PND) 180, but not at PND90. We found that serum level of luteinizing hormone (LH) at the latter half of proestrus in EE-treated rats was lower than in the controls at PND90 when there was no significant difference on estrous cyclicity. Additionally, kiss1 mRNA levels in the anteroventral periventricular nucleus-preoptic area (AVPV/POA) were lower in EE-treated rats than in the controls. The expression of GnRH precursor (GNRH1) mRNA in the AVPV/POA and that of LH beta subunit (LHb) mRNA in the pituitary were similar in the control- and EE-treated groups. Our results indicated that neonatal exposure to EE leads to reduced expression of kiss1 mRNA in AVPV/POA and LH-surge, which is likely related to the delayed reproductive dysfunction seen in adult female rats.
PMCID: PMC4155190  PMID: 24784441
17α-ethinyl estradiol; AVPV/POA; endocrine disruptor; kisspeptin
15.  Impact of neonatal exposure to the ERα agonist PPT, bisphenol-a or phytoestrogens on hypothalamic kisspeptin fiber density in male and female rats 
Neurotoxicology  2009;30(3):350-357.
Neonatal exposure to endocrine disrupting compounds (EDCs) can impair reproductive physiology, but the specific mechanisms by which this occurs remain largely unknown. Growing evidence suggests that kisspeptin (KISS) neurons play a significant role in the regulation of pubertal onset and ovulation, therefore disruption of KISS signaling could be a mechanism by which EDCs impair reproductive maturation and function. We have previously demonstrated that neonatal exposure to phytoestrogens decreases KISS fiber density in the anterior hypothalamus of female rats, an effect which was associated with early persistent estrus and the impaired activation gonadotropin releasing hormone (GnRH) neurons. The goals of the present study were to (1) determine if an ERα selective agonist (PPT) or bisphenol-A (BPA) could produce similar effects on hypothalamic KISS content in female rats and (2) to determine if male KISS fiber density was also vulnerable to disruption by EDCs. We first examined the effects of neonatal exposure to PPT, a low (50 μg/kg bw) BPA dose, and a high (50 mg/kg bw) BPA dose on KISS immunoreactivity (-ir) in the anterior ventral periventricular (AVPV) and arcuate (ARC) nuclei of adult female rats, using estradiol benzoate (EB) and a sesame oil vehicle as controls. AVPV KISS-ir, following ovariectomy (OVX) and hormone priming, was significantly lower in the EB and PPT groups but not the BPA groups. ARC KISS-ir levels were significantly diminished in the EB and high dose BPA groups, and there was a nonsignificant trend for lower KISS-ir in the PPT group. We next examined effects of neonatal exposure to a low (50μg/kg bw) dose of BPA and the phytoestrogens genistein (GEN) and equol (EQ) on KISS-ir in the AVPV and ARC of adult male rats, using OVX females as an additional control group. None of the compounds affected KISS-ir in the male hypothalamus. Our results suggest that the organization of hypothalamic KISS fibers may be vulnerable to disruption by EDC exposure and that females might be more sensitive than males.
PMCID: PMC3691558  PMID: 19442818
bisphenol; phytoestrogens; soy; genistein; equol; gonadotropins; development; disruption; estrogen; estrogen receptor; GnRH; KISS
16.  17β-Estradiol and Progesterone Regulate Multiple Progestin Signaling Molecules in the Anteroventral Periventricular Nucleus, Ventromedial Nucleus and Sexually Dimorphic Nucleus of the Preoptic Area in Female Rats 
Neuroscience  2010;176:86-92.
Recent work identified novel progestin signaling molecules, including progesterone receptor membrane component 1 (Pgrmc1), Pgrmc2, serpine mRNA binding protein 1 (Serbp1), progestin and adiponectin receptors 7 (Paqr7) and Paqr8. These molecules mediate rapid progesterone (P4) effects in non-neural tissue and we recently mapped their expression in the brain. Many rapid effects of P4 require 17β-estradiol (E2) and P4 priming; therefore, we examined the effects of ovarian hormones on the expression of these non-classical progestin signaling molecules. We focused specifically on the anteroventral periventricular nucleus (AVPV), the sexually dimorphic nucleus of the preoptic area (SDN-POA) and the ventrolateral portion of the ventromedial nucleus (VMNvl). These brain nuclei are important for female reproduction. Ovariectomized adult female rats were implanted with capsules containing sesame oil or E2, and injected 48 hours later with sesame oil or P4. Brains were collected eight hours later and RNA was isolated from the AVPV, SDN-POA and VMNvl. We assessed the effects of ovarian hormones on mRNA levels using quantitative polymerase chain reaction (QPCR). In the AVPV, Serbp1 mRNA levels were increased by P4 in the presence of E2, and Paqr8 was downregulated by P4 alone. In the SDN-POA, combined E2 and P4 increased Pgrmc1 and Serbp1 mRNA levels, and E2 alone increased Paqr8 mRNA levels. Finally, in the VMNvl, P4 increased mRNA levels encoding Pgrmc1, Pgrmc2 and Serbp1, and the combination of E2 and P4 increased Pgrmc1 and Serbp1 mRNA levels. Paqr7 was not regulated by E2 or P4 in any brain region examined. In summary, we showed that ovarian hormones regulate novel progestin signaling molecules in brain regions important for the neuroendocrine control of reproduction.
PMCID: PMC3046387  PMID: 21185909
Estradiol; progesterone; neuroendocrine; non-classical signaling; receptor
17.  Reproductive Hormone-Dependent and -Independent Contributions to Developmental Changes in Kisspeptin in GnRH-Deficient Hypogonadal Mice 
PLoS ONE  2010;5(7):e11911.
Kisspeptin is a potent activator of GnRH-induced gonadotropin secretion and is a proposed central regulator of pubertal onset. In mice, there is a neuroanatomical separation of two discrete kisspeptin neuronal populations, which are sexually dimorphic and are believed to make distinct contributions to reproductive physiology. Within these kisspeptin neuron populations, Kiss1 expression is directly regulated by sex hormones, thereby confounding the roles of sex differences and early activational events that drive the establishment of kisspeptin neurons. In order to better understand sex steroid hormone-dependent and -independent effects on the maturation of kisspeptin neurons, hypogonadal (hpg) mice deficient in GnRH and its downstream effectors were used to determine changes in the developmental kisspeptin expression. In hpg mice, sex differences in Kiss1 mRNA levels and kisspeptin immunoreactivity, typically present at 30 days of age, were absent in the anteroventral periventricular nucleus (AVPV). Although immunoreactive kisspeptin increased from 10 to 30 days of age to levels intermediate between wild type (WT) females and males, corresponding increases in Kiss1 mRNA were not detected. In contrast, the hpg arcuate nucleus (ARC) demonstrated a 10-fold increase in Kiss1 mRNA between 10 and 30 days in both females and males, suggesting that the ARC is a significant center for sex steroid-independent pubertal kisspeptin expression. Interestingly, the normal positive feedback response of AVPV kisspeptin neurons to estrogen observed in WT mice was lost in hpg females, suggesting that exposure to reproductive hormones during development may contribute to the establishment of the ovulatory gonadotropin surge mechanism. Overall, these studies suggest that the onset of pubertal kisspeptin expression is not dependent on reproductive hormones, but that gonadal sex steroids critically shape the hypothalamic kisspeptin neuronal subpopulations to make distinct contributions to the activation and control of the reproductive hormone cascade at the time of puberty.
PMCID: PMC2912854  PMID: 20689830
18.  Oestrogen-independent circadian clock gene expression in the anteroventral periventricular nucleus in female rats: Possible role as an integrator for circadian and ovarian signals timing the LH surge 
Journal of neuroendocrinology  2013;25(12):1273-1279.
Periodic ovulation in rats, mice and hamsters is the result of a surge in LH that depends on circadian gating signals emerging from the master circadian clock within the suprachiasmatic nucleus (SCN) and rising ovarian oestrogen levels. These two signals converge into the anteroventral periventricular nucleus (AVPV) and lead to the release of kisspeptin, which is responsible for surges of GnRH and, in turn, of LH release. How the AVPV integrates circadian and reproductive signals remains unclear. Here we show that the female rat AVPV itself shows circadian oscillations in the expression of the clock genes PER1 and BMAL1, which lie at the core circadian clockwork of mammals. In ovariectomized (OVX) females treated with estradiol (E2) these oscillations are in synchrony with the AVPV rhythmic expression of the KISS1 gene and the gene that codes for the arginine-vasopressin (AVP) receptor AVPr1a. Whereas clock gene oscillations are independent of oestrogen levels, circadian expression of Kiss1 and Avpr1a (also referred to as V1a) mRNA are respectively blunted and absent in ovariectomized animals without E2 replacement. Because AVP is believed to be a critical SCN transmitter to gate the LH surge, our data suggest that a there is a circadian oscillator located in the AVPV, and that such a putative oscillator could time, in an oestrogen dependent manner, the sensitivity to circadian signals emerging from the SCN and the release of kisspeptin.
PMCID: PMC3954464  PMID: 24028332
kisspeptin; vasopressin; circadian oscillators; ovulation
19.  Regional Difference in Sex Steroid Action on Formation of Morphological Sex Differences in the Anteroventral Periventricular Nucleus and Principal Nucleus of the Bed Nucleus of the Stria Terminalis 
PLoS ONE  2014;9(11):e112616.
Sex steroid action is critical to form sexually dimorphic nuclei, although it is not fully understood. We previously reported that masculinization of the principal nucleus of the bed nucleus of the stria terminalis (BNSTp), which is larger and has more neurons in males than in females, involves aromatized testosterone that acts via estrogen receptor-α (ERα), but not estrogen receptor-β (ERβ). Here, we examined sex steroid action on the formation of the anteroventral periventricular nucleus (AVPV) that is larger and has more neurons in females. Morphometrical analysis of transgenic mice lacking aromatase, ERα, or ERβ genes revealed that the volume and neuron number of the male AVPV were significantly increased by deletion of aromatase and ERα genes, but not the ERβ gene. We further examined the AVPV and BNSTp of androgen receptor knockout (ARKO) mice. The volume and neuron number of the male BNSTp were smaller in ARKO mice than those in wild-type mice, while no significant effect of ARKO was found on the AVPV and female BNSTp. We also examined aromatase, ERα, and AR mRNA levels in the AVPV and BNSTp of wild-type and ARKO mice on embryonic day (ED) 18 and postnatal day (PD) 4. AR mRNA in the BNSTp and AVPV of wild-type mice was not expressed on ED18 and emerged on PD4. In the AVPV, the aromatase mRNA level was higher on ED18, although the ERα mRNA level was higher on PD4 without any effect of AR gene deletion. Aromatase and ERα mRNA levels in the male BNSTp were significantly increased on PD4 by AR gene deletion. These results suggest that estradiol signaling via ERα during the perinatal period and testosterone signaling via AR during the postnatal period are required for masculinization of the BNSTp, whereas the former is sufficient to defeminize the AVPV.
PMCID: PMC4232352  PMID: 25398007
20.  Characterization of Kiss1 neurons using transgenic mouse models 
Neuroscience  2010;173:37-56.
Humans and mice with loss-of-function mutations of the genes encoding kisspeptins (Kiss1) or kisspeptin receptors (Kiss1r) are infertile due to hypogonadotropic hypogonadism. Within the hypothalamus, Kiss1 mRNA is expressed in the anteroventral periventricular nucleus (AVPV) and the arcuate nucleus (Arc). In order to better study the different populations of kisspeptin cells we generated Kiss1-Cre transgenic mice. We obtained one line with Cre activity specifically within Kiss1 neurons (line J2-4), as assessed by generating mice with Cre-dependent expression of green fluorescent protein or β-galactosidase. Also, we demonstrated Kiss1 expression in the cerebral cortex and confirmed previous data showing Kiss1 mRNA in the medial nucleus of amygdala and anterodorsal preoptic nucleus. Kiss1 neurons were more concentrated towards the caudal levels of the Arc and higher leptin-responsivity was observed in the most caudal population of Arc Kiss1 neurons. No evidence for direct action of leptin in AVPV Kiss1 neurons was observed. Melanocortin fibers innervated subsets of Kiss1 neurons of the preoptic area and Arc, and both populations expressed MC4R. Specifically in the preoptic area, 18–28% of Kiss1 neurons expressed MC4R. In the Arc, 90% of Kiss1 neurons were glutamatergic, 50% of which also were GABAergic. In the AVPV, 20% of Kiss1 neurons were glutamatergic whereas 75% were GABAergic. The differences observed between the Kiss1 neurons in the preoptic area and the Arc likely represent neuronal evidence for their differential roles in metabolism and reproduction.
PMCID: PMC3026459  PMID: 21093546
leptin; melanocortin; Cre-recombinase; reporter mouse; GAD-67; vGluT2
Neurotoxicology  2008;29(6):988-997.
It is well established that estrogen administration during neonatal development can advance pubertal onset and prevent the maintenance of regular estrous cycles in female rats. This treatment paradigm also eliminates the preovulatory rise of gonadotropin releasing hormone (GnRH). It remains unclear, however, through which of the two primary forms of the estrogen receptor (ERα or ERβ) this effect is mediated. It is also unclear whether endocrine disrupting compounds (EDCs) can produce similar effects. Here we compared the effect of neonatal exposure to estradiol benzoate (EB), the ERα specific agonist 1,3,5-tris(4-Hydroxyphenyl)-4-propyl-1H-pyrazole (PPT), the ERβ specific agonist diarylpropionitrile (DPN) and the naturally occurring EDCs genistein (GEN) and equol (EQ) on pubertal onset, estrous cyclicity, GnRH activation, and kisspeptin content in the anteroventral periventricular (AVPV) and arcuate (ARC) nuclei. Vaginal opening was significantly advanced by EB and GEN. By ten weeks post-puberty, irregular estrous cycles were observed in all groups except the control group. GnRH activation, as measured by the percentage of immunopositive GnRH neurons that were also immunopositive for Fos, was significantly lower in all treatment groups except the DPN group compared to the control group. GnRH activation was absent in the PPT group. These data suggest that neonatal exposure to EDCs can suppress GnRH activity in adulthood, and that ERα plays a pivotal role in this process. Kisspeptins (KISS) have recently been characterized to be potent stimulators of GnRH secretion. Therefore we quantified the density of KISS immunolabeled fibers in the AVPV and ARC. In the AVPV, KISS fiber density was significantly lower in the EB and GEN groups compared to the control group but only in the EB and PPT groups in the ARC. The data suggest that decreased stimulation of GnRH neurons by KISS could be a mechanism by which EDCs can impair female reproductive function.
PMCID: PMC2647326  PMID: 18656497
genistein; equol; soy; isoflavones; gonadotropin; puberty; development; endocrine disruption; DPN; PPT
22.  Quantification of Rat Kisspeptin Using a Novel Radioimmunoassay 
PLoS ONE  2014;9(5):e97611.
Kisspeptin is a hypothalamic peptide hormone that plays a pivotal role in pubertal onset and reproductive function. Previous studies have examined hypothalamic kisspeptin mRNA expression, either through in situ hybridisation or real-time RT-PCR, as a means quantifying kisspeptin gene expression. However, mRNA expression levels are not always reflected in levels of the translated protein. Kisspeptin-immunoreactivity (IR) has been extensively examined using immunohistochemistry, enabling detection and localisation of kisspeptin perikaya in the arcuate nucleus (ARC) and anteroventral periventricular nucleus (AVPV). However, quantification of kisspeptin-IR remains challenging. We developed a specific rodent radioimmunoassay assay (RIA) capable of detecting and quantifying kisspeptin-IR in rodent tissues. The RIA uses kisspeptin-10 as a standard and radioactive tracer, combined with a commercially available antibody raised to the kisspeptin-10 fragment. Adult female wistar rat brain samples were sectioned at 300 µm and the ARC and AVPV punch micro-dissected. Brain punches were homogenised in extraction buffer and assayed with rodent kisspeptin-RIA. In accord with the pattern of kisspeptin mRNA expression, kisspeptin-IR was detected in both the ARC (47.1±6.2 fmol/punch, mean±SEM n = 15) and AVPV (7.6±1.3 fmol/punch, mean±SEM n = 15). Kisspeptin-IR was also detectable in rat placenta (1.26±0.15 fmol/mg). Reverse phase high pressure liquid chromatography analysis showed that hypothalamic kisspeptin-IR had the same elution profile as a synthetic rodent kisspeptin standard. A specific rodent kisspeptin-RIA will allow accurate quantification of kisspeptin peptide levels within specific tissues in rodent experimental models.
PMCID: PMC4028310  PMID: 24845101
23.  Sexual differentiation and the Kiss1 system: hormonal and developmental considerations 
Peptides  2008;30(1):83-93.
The nervous system (both central and peripheral) is anatomically and physiologically differentiated between the sexes, ranging from gender-based differences in the cerebral cortex to motoneuron number in the spinal cord. Although genetic factors may play a role in the development of some sexually differentiated traits, most identified sex differences in the brain and behavior are produced under the influence of perinatal sex steroid signaling. In many species, the ability to display an estrogen-induced luteinizing hormone (LH) surge is sexually differentiated, yet the specific neural population(s) that allows females but not males to display such estrogen-mediated “positive feedback” has remained elusive. Recently, the Kiss1/kisspeptin system has been implicated in generating the sexually-dimorphic circuitry underlying the LH surge. Specifically, Kiss1 gene expression and kisspeptin protein levels in the anteroventral periventricular (AVPV) nucleus of the hypothalamus are sexually differentiated, with females displaying higher levels than males, even under identical hormonal conditions as adults. These findings, in conjunction with accumulating evidence implicating kisspeptins as potent secretagogues of gonadotropin-releasing hormone (GnRH), suggest that the sex-specific display of the LH surge (positive feedback) reflects sexual differentiation of AVPV Kiss1 neurons. In addition, developmental kisspeptin signaling via its receptor GPR54 appears to be critical in males for the proper sexual differentiation of a variety of sexually dimorphic traits, ranging from complex social behavior to specific forebrain and spinal cord neuronal populations. This review discusses the recent data, and their implications, regarding the bidirectional relationship between the Kiss1 system and the process of sexual differentiation.
PMCID: PMC2631352  PMID: 18644414
metastin; kisspeptin; Kiss1; GPR54; sexual differentiation; sex differences; AVPV; tyrosine hydroxylase; development
24.  Gonadal and Non-Gonadal Regulation of Sex Differences in Hypothalamic Kiss1 Neurons 
Journal of neuroendocrinology  2010;22(7):682-691.
The brains of males and females differ anatomically and physiologically, including sex differences in neuron size or number, synapse morphology, and specific patterns of gene expression. Brain sex differences may underlie critical sex differences in physiology or behavior, including several aspects of reproduction, such as the timing of sexual maturation (earlier in females than males) and the ability to generate a preovulatory gonadotropin surge (in females only). The reproductive axis is controlled by afferent pathways that converge upon forebrain gonadotropin-releasing hormone (GnRH) neurons, but GnRH neurons are not sexually dimorphic. Although most reproductive sex differences probably reflect sex differences in the upstream circuits and factors that regulate GnRH secretion, the key sexually-dimorphic factors that influence reproductive status have remained poorly defined. The recently-identified neuropeptide kisspeptin, encoded by the Kiss1 gene, is an important regulator of GnRH secretion, and Kiss1 neurons in rodents are sexually dimorphic in specific hypothalamic populations, including the anteroventral periventricular nucleus—periventricular nucleus continuum (AVPV/PeN) and the arcuate nucleus (ARC). In the adult AVPV/PeN, Kiss1 neurons are more abundant in females than males, a sex difference which is regulated by estradiol signaling during critical periods of postnatal and pubertal development. In contrast, Kiss1 neurons in the ARC are not sexually differentiated in adult rodents, but in mice, the regulation of ARC Kiss1 cells by gonadal hormone-independent factors is sexually dimorphic during prepubertal development. These various sex differences in hypothalamic Kiss1 neurons may relate to known sex differences in reproductive physiology, such as puberty onset and positive feedback.
PMCID: PMC3096441  PMID: 20492362
kisspeptin; Kiss1; GPR54; Kiss1r; sexual differentiation; sex differences; development; puberty; hypothalamus; hormone; estrogen
25.  The NMDA Receptor Subunit NR2b: Effects on LH Release and GnRH Gene Expression in Young and Middle-aged Female Rats, with Modulation by Estradiol 
Neuroendocrinology  2007;87(3):129-141.
The loss of reproductive capacity during aging involves changes in the neural regulation of the hypothalamic gonadotropin-releasing hormone (GnRH) neurons controlling reproduction. This neuronal circuitry includes glutamate receptors on GnRH neurons. Previously, we reported an increase in the expression of the NR2b subunit protein of the NMDA receptor on GnRH neurons in middle-aged compared to young female rats. Here, we examined the functional implications of the NR2b subunit on the onset of reproductive aging, using an NR2b-specific antagonist ifenprodil. Young (3–5 mos.) and middle-aged (10–13 mos.) female rats were ovariectomized (OVX), 17β-estradiol (E2) or vehicle (cholesterol) treated, and implanted with a jugular catheter. Serial blood sampling was undertaken every 10 minutes for 4 hours, with ifenprodil (10mg/kg) or vehicle injected (i.p.) after one hour of baseline sampling. The pulsatile release of pituitary LH and levels of GnRH mRNA in hypothalamus were quantified as indices of the reproductive axis. Our results showed effects of ifenprodil on both endpoints. In OVX rats given cholesterol, neither age nor ifenprodil had any effects on LH release. In E2-treated rats, aging was associated with significant decreases in pulsatile LH release. Additionally, ifenprodil stimulated parameters of pulsatile LH release in both young and middle-aged animals. Ifenprodil had few effects on GnRH mRNA; the only significant effect of ifenprodil was found in the middle-aged, cholesterol group. Together, these findings support a role for the NR2b subunit of the NMDAR in GnRH/LH regulation. Because most of these effects were exhibited on pituitary LH release in the absence of a concomitant change in GnRH gene expression, it is likely that NMDA receptors containing the NR2b subunit plays a role in GnRH-induced LH release, independent of de novo GnRH gene expression.
PMCID: PMC2671961  PMID: 18025808
Luteinizing hormone (LH); gonadotropin-releasing hormone (GnRH); glutamate; N-methyl-D-Aspartate receptor (NMDAR); NR2b; reproductive aging; reproductive senescence; estrogen; GnRH mRNA; ifenprodil

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