Immunoelectron microscopic observation of the subcellular localization of kisspeptin, neurokinin B and dynorphin A in KNDy neurons in the arcuate nucleus of the female rat

Highlights

• ARC kisspeptin neurons co-express with NKB and DynA.

• Subcellular localization of kisspeptin, NKB and DynA in KNDy neurons were observed by immunoelectron microscopy.

• Kisspeptin, NKB and DynA are contained within separate individual neurosecretory vesicles.

Abstract

KNDy neurons are named for their co-expression of three neuropeptides, kisspeptin, neurokinin B (NKB) and dynorphin A (DynA). These cells, located in the hypothalamic arcuate nucleus (ARC), are associated with generation of gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH) pulses to control follicular growth and steroidogenesis. However, subcellular sorting mechanisms for secretory vesicles containing these neuropeptides have not been elucidated. In this study, we analyzed the localization pattern of kisspeptin, NKB and DynA in the ARC of the ovariectomized rat immediately treated with estrogen using immunoelectron microscopy. First, we identified neuropeptides by dual-labeled fluorescence immunohistochemistry, with results indicating all three neuropeptides co-express within individual ARC cells in female rats. Next, we investigated the subcellular localization pattern of kisspeptin, NKB, and/or DynA using post-embedding double immunoelectron microscopy, indicating that each type of neuropeptide is contained within separate and individual neurosecretory vesicles. This suggests sorting and packaging of kisspeptin, NKB and DynA is differentially regulated within KNDy neurons. Our findings facilitate understanding of regulatory mechanisms underlying kisspeptin secretion in KNDy neurons, and generation of GnRH/LH pulses induced by kisspeptin in the ARC.

Introduction

Gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH) pulses are necessary for reproduction [1]. Abnormal LH pulses have been observed in women with polycystic ovary syndrome (PCOS) [2] and weight loss-related amenorrhea [3]. Thus, GnRH/LH pulse abnormalities have the potential to cause menstrual disorders, anovulation and infertility.

Recent studies suggest pulsatile GnRH/LH secretion is generated by KNDy neurons [4]. These cells, which co-express kisspeptin, neurokinin B (NKB) and dynorphin A (DynA), have been identified in the hypothalamic arcuate nucleus (ARC) of mice [5], rats [6], ewes [7] and goats [8]. Kisspeptin fibers project, in a non-synaptic manner, to GnRH terminals located within the median eminence (ME) of monkeys [9], rats and goats [10], [11]. GnRH neurons expressing kisspeptin receptors (Kiss1r) have been identified in mice [12] and rats [13]. KNDy neurons also express estrogen receptor α (ERα) in mice [14], rats [6], [15] and ewes [16], and receive to negative feedback of estrogen to regulate LH release [14], [15].

NKB also induces LH release, as evidenced by the ability of a selective NKB receptor (NK3R) agonist, senktide, to increase LH secretion in sheep [17] and multiple-unit electrical activity (MUA) volleys in the ARC of goats [8]. In contrast, DynA inhibits LH release, as demonstrated by the capacity of a selective DynA receptor (KOR) antagonist, nor-binaltorphimine (nor-BNI), to increase LH pulse frequency in sheep [18]. Further, MUA volleys in the ARC are inhibited by DynA and increased by nor-BNI in goats [8].

As KNDy fibers have been observed in close apposition to KNDy cell bodies in the ARC of rats [6], ovine [19] and goats [8], it is likely KNDy neurons communicate with each other. KNDy neurons express NK3R in mice [5], rats [6], [20], and sheep [21]; KOR in mice [5]; but, do not express Kiss1r in mice [22] and ovine [23]. In rats, GnRH neurons do not express KOR and only slightly express NK3R [20], [24]; while in sheep, these cells do not express NK3R [21]. Further, as senktide was shown to have no effect on GnRH neuronal firing in castrated male mice [25], GnRH neurons appear to be unaffected by NKB or DynA. Thus, KNDy neurons are thought to act as GnRH/LH pulse generators within neural circuits by regulating kisspeptin secretion through stimulation by NKB/NK3R signaling and inhibition by DynA/KOR signaling.

Though co-expression of kisspeptin, NKB and DynA has previously been reported in mice [5], ewes [7] and goats [8], studies examining rat KNDy neurons indicate only proNKB and prodynorphin co-localize within ARC kisspeptin neurons [6]. Moreover, subcellular localization of neuropeptides within these cells has not been elucidated. Thus, in the current study, we investigated the subcellular localization of kisspeptin, NKB and DynA within KNDy neurons of female rats by double immunoelectron microscopy.