Immunohistochemical analysis of the colocalization of corticotropin-releasing hormone receptor and glucocorticoid receptor in kisspeptin neurons in the hypothalamus of female rats
Highlights
► Almost all kisspeptin neurons in both of AVPV/PeN and ARC expressed CRH-R. ► A few close appositions of CRH fibers on some of kisspeptin neurons were observed. ► Many kisspeptin neurons in AVPV/PeN expressed GR. ► On the other hand, only a few kisspeptin neurons in ARC expressed GR.
Introduction
Stress has been known to suppress reproductive function. Various stressors such as inflammatory signals [29], physical restraint [31] and insulin-induced hypoglycemia [4] profoundly suppress the pulsatile secretion of luteinizing hormone (LH) from the anterior pituitary. It is well established that the inhibitory effect of stress on gonadotropin secretion is mediated by corticotropin-releasing hormone (CRH) and glucocorticoids, which are the molecules induced by many types of stressors. Central administration of CRH results in a rapid and dose-dependent inhibition of LH secretion [28], [30], [41]. Conversely, stress-induced suppression of LH secretion can be rescued by CRH antagonist [7], [39]. Although glucocorticoid has no acute effect on LH secretion [23], chronic administration is associated with a decrease in the circulating LH level [3], [13].
Kisspeptin, a neuropeptide encoded by Kiss1 gene, has been shown to play important roles in the development and regulation of reproductive functions. Administration of kisspeptin revealed that kisspeptin is a strong secretagogue of gonadotropin and its stimulatory effect is mediated via gonadotropin-releasing hormone (GnRH) neuron [16], [19], [25], [32]. Kisspeptin neurons have been known to localize mainly in the anteroventral periventricular nucleus and periventricular nucleus continuum (AVPV/PeN) and in the arcuate nucleus (ARC) in the rodent hypothalamus [16], [33], [34]. These two populations of kisspeptin neurons are implicated to be involved in the gonadal steroid feedback regulation of gonadotropin release, since kisspeptin neurons express estrogen receptor α and the expression of Kiss1 mRNA in AVPV/PeN and ARC is regulated by gonadal steroids [1], [33], [34], [37]. It is known that there are sex differences in the expressions of kisspeptin. In AVPV/PeN of adult rodents, Kiss1 mRNA expression and the number of kisspeptin immunoreactive neurons are greater in females than in males [1], [10], [21]. In addition, kisspeptin immunoreactivity in ARC of adult rats is also greater in females [12], [18], although Kiss1 mRNA are comparable between sexes [21].
Recently, it has been reported that stressors, such as restraint and immune challenge, decrease the Kiss1 level in the hypothalamus along with reduction of LH secretion [22], indicating that the suppression of LH secretion under stress is mediated via the kisspeptin system. Furthermore, administration of CRH or corticosterone decreases Kiss1 expression in both AVPV/PeN and ARC [22]. However, whether those stress-related molecules can directly act on kisspeptin neurons remains unknown.
CRH acts via interaction with CRH receptor (CRH-R), which is a G protein-coupled receptor. Two CRH-R subtypes, type 1 (CRH-R1) and type 2 (CRH-R2), have been identified and the expression patterns and physiological functions are known to be different between subtypes [6], [14], [40]. As for corticosteroids, two types of receptor, mineralcorticoid receptor (MR) and glucocorticoid receptor (GR), exist in the brain. The effects of elevated circulating glucocorticoids in response to stress are believed to be mediated by GR because of the difference in the affinities for glucocorticoids between MR and GR [11], [15].
In this study, we performed double fluorescence immunohistochemistry to determine whether kisspeptin neurons in rat hypothalamus express CRH-R and GR. We also analyzed the spatial relationship between kisspeptin neurons and CRH immunoreactive fibers.
Section snippets
Animals and treatments
Adult female Wistar rats (8–10 weeks of age, n = 10) were purchased from Saitama Experimental Animal Supply Co., Ltd. (Saitama, Japan) and kept under controlled condition of light (14 h light/day; lights on from 6:00) and temperature (24 ± 2 °C) with free access to standard rodent chow and water. The stage of estrus cycle was determined by daily vaginal smear examination, and rats that showed at least two consecutive 4 or 5-day estrus cycle were used. A group of female rats (n = 5) in the second day of
Results
We observed CRH-R immunoreactivity throughout the hypothalamus and particularly in AVPV/PeN and ARC. Fig. 1 shows representative photographs of CRH-R immunoreactivity in kisspeptin immunoreactive neurons in AVPV/PeN and ARC. Clear CRH-R immunoreactivity was observed in the cell body of kisspeptin immunoreactive neurons but not in the fibers. Quantitative analysis revealed that almost all kisspeptin immunoreactive neurons in both of AVPV/PeN and ARC showed CRH-R immunoreactivity. The percentages
Discussion
It is well established that stress inhibits reproductive function. Several stress-related molecules have been shown to be involved in the suppression of gonadotropin secretion under stress. In the present study, we explored the possibility that CRH and glucocorticoids directly act on kisspeptin neurons, which are an important regulator of gonadotropin secretion, by determining the expression of CRH-R and GR in kisspeptin neurons in female rat hypothalamus. Since kisspeptin immunoreactivity in
Acknowledgements
We are grateful to Takeda Pharmaceutical Company, Ltd. for kindly providing mouse anti-kisspeptin antibody, and to Prof. Kawata, Kyoto Prefectural University of Medicine, for providing rabbit anti-GR antibody. This work was supported by Grants-in-Aid from Ministry of Education, Science, Sports and Culture, Japan (#S0801035, 22590230 to H.O. and 23659125 to N. I.), and the MEXT-Supported Program for the Strategic Research Foundation at Private Universities.
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Heterogeneity in GnRH and kisspeptin neurons and their significance in vertebrate reproductive biology
2022, Frontiers in NeuroendocrinologyCitation Excerpt :Under any form of stress, the hypothalamus–pituitaryadrenal (HPA) axis is triggered and stress hormones such as corticotropin-releasing hormone (CRH) and glucocorticoid are released. CRH and glucocorticoid directly acts on GnRH and kisspeptin neurons (Attardi et al., 1997; Chandran et al., 1994, Chandran et al., 1996; Grachev et al., 2013, Luo et al., 2016, Raftogianni et al., 2018, Takumi et al., 2012; Tilbrook et al., 2000). A number of metabolic factors such as leptin and insulin are also known to act on GnRH and kisspeptin neurons (Castellano and Tena-Sempere, 2013, Gamba and Pralong, 2006).
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2021, Frontiers in NeuroendocrinologyCitation Excerpt :Ascribing these effects to direct action on kisspeptin neurons is tempting but somewhat tenuous for at least two reasons: controversy over expression of appropriate receptors by these cells and the difference in time course of the suppression of LH pulses vs the immunohistochemical measures. With regard to receptors for HPA axis hormones, immunoreactive glucocorticoid receptors have been reported in arcuate kisspeptin neurons of both OVX and OVX + E mice (Kreisman et al., 2019), but another study identified these receptors in only AVPV kisspeptin neurons (Takumi et al., 2012). As with GnRH neurons, expression of CRHRs by kisspeptin neurons is unresolved.
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2021, Free Radical Biology and MedicineCitation Excerpt :Previous studies have reported the E2-dependent hypersecretion of glucocorticoids [62,63]. Moreover, stress-induced glucocorticoid production-suppressed kisspeptin mRNA expression has been proven [64], and in the arcuate nucleus chronic corticosterone reduces kisspeptin neuronal activity through an E2-dependent machinery [65]. Therefore, this study assumes that E2 inhibits the FSH and LH secretion from the sertoli and leydig cells via the enhancement of corticosterone secretion, which further impairs the kisspeptin/GnRH signaling cascade.
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