Regulation of GnRH pulsatility in ewes

Abstract

Early work in ewes provided a wealth of information on the physiological regulation of pulsatile gonadotropin-releasing hormone (GnRH) secretion by internal and external inputs. Identification of the neural systems involved, however, was limited by the lack of information on neural mechanisms underlying generation of GnRH pulses. Over the last decade, considerable evidence supported the hypothesis that a group of neurons in the arcuate nucleus that contain kisspeptin, neurokinin B and dynorphin (KNDy neurons) are responsible for synchronizing secretion of GnRH during each pulse in ewes. In this review, we describe our current understanding of the neural systems mediating the actions of ovarian steroids and three external inputs on GnRH pulsatility in light of the hypothesis that KNDy neurons play a key role in GnRH pulse generation. In breeding season adults, estradiol (E₂) and progesterone decrease GnRH pulse amplitude and frequency, respectively, by actions on KNDy neurons, with E₂ decreasing kisspeptin and progesterone increasing dynorphin release onto GnRH neurons. In pre-pubertal lambs, E₂ inhibits GnRH pulse frequency by decreasing kisspeptin and increasing dynorphin release, actions that wane as the lamb matures to allow increased pulsatile GnRH secretion at puberty. Less is known about mediators of undernutrition and stress, although some evidence implicates kisspeptin and dynorphin, respectively, in the inhibition of GnRH pulse frequency by these factors. During the anoestrus, inhibitory photoperiod acting via melatonin activates A15 dopaminergic neurons that innervate KNDy neurons; E₂ increases dopamine release from these neurons to inhibit KNDy neurons and suppress the frequency of kisspeptin and GnRH release.

Figure 1

Model for control of KNDy neural activity proposed to drive episodic GnRH secretion. Each GnRH pulse is initiated by NKB (purple) acting within the KNDy network (within dashed oval), which stimulates kisspeptin (green) release to drive GnRH (blue) secretion and activate unidentified Kiss1r-containing ARC neurons (grey) that reinforces the stimulatory actions of NKB on KNDy neurons. GnRH release is then terminated by dynorphin (red) release from KNDy neurons acting either directly on KNDy neurons and/or GnRH neurons and/or the unidentified Kiss1r-containing neurons. Abbreviations: R_dyn: kappa-opioid receptor; R_Ks: Kiss1r; R_NKB: NK3R. Note that the color in each terminal indicates the biologically active transmitter (possibly due to selective expression of post-synaptic receptors) and does not reflect selective transport of that peptide to the terminal. Redrawn from Lehman et al., (8) with permission from the Endocrine Society.

Figure 2

Receptors for KNDy peptides in GnRH and kisspeptin neurons. Top four panels depict dual ICC for NK3R (red in A, green in C) and KOR (red in B and D) in GnRH neurons (green in A and B) and kisspeptin neurons (red in C, green in D). Arrows identify dual labeled cells, arrowheads indicate single labeled KOR neurons. Bottom panels present the mean (± SEM) percentage of GnRH and kisspeptin cells in the POA and MBH (KNDy cells for latter) containing each receptor. Note no GnRH cells contained NK3R. NA: Not analyzed. Photomicrographs in Panels A and C reprinted from Amstalden et al., 2010 and Ahn et al., J Neuroendocrinol 27: 100–110, 2015, respectively with permission from the British Society for Neuroendocrinology. Photomicrographs in panels B and D reprinted from Weems et al. 2016, with permission from the Endocrine Society. Bar graphs in bottom panel are based on data from these three references (NK3R and KOR) and from Smith et al, 2011 (Kiss1r).

Figure 3

Effect of a KOR antagonist on LH pulse frequency in pre- and post-pubertal lambs. Top panels: LH pulse patterns in response to lateral ventricle infusions of vehicle (Control) or a KOR antagonist (BNI) in OVX+E pre-pubertal (Panels A,B) and post-pubertal (Panels C,D) lambs. *Peak of LH pulse. Bottom panels: Mean (± SEM) LH pulse frequency in response to lateral ventricle infusions of vehicle (Cont) or the KOR antagonist (BNI) in oestrogen-treated OVX pre- and post-pubertal lambs are shown on left (Panel E). Effect of empty (Cont) or BNI-filled microimplants in the ARC or POA of ovary-intact pre-pubertal lambs on pulse frequency are depicted on right (Panel F).*P<0.05 vs Cont. Data on lateral ventricle infusions redrawn from Lopez et al., 2016 with permission from the British Society for Neuroendocrinology. Data on the effects of microimplants have not been published.

Figure 4

Evidence for possible roles of each KNDy peptide in mediating E₂ negative feedback in breeding season ewes. Top panels: Effect of an EOP receptor antagonist on mean (± SEM) LH (A) and GnRH (B) pulse amplitude in OVX and OVX+E breeding season ewes. * P<0.05 vs pre-treatment values. Bottom panel (C): Effect of E₂ on the mean (± SEM) number of cells in the ARC containing kisspeptin, NKB, or dynorphin in OVX breeding season ewes. *P<0.05 vs OVX. LH data reprinted from Whisnant and Goodman, Biol Reprod 39: 1032–38, 1988 with permission from the Society for the Study of Reproduction. GnRH and KNDy peptide data reprinted from Goodman et al., 1995 and Weems et al., 2017a, respectively, with permission of the Endocrine Society.

Figure 5

Role of KNDy neurons in the control of pulsatile GnRH secretion in ewes under different physiological conditions. In well-fed prepubertal lambs (top left panel), GnRH pulse frequency is held in check by E₂-induced decreases in kisspeptin and increases in dynorphin release, but this could be due to either direct or indirect actions of E₂ on KNDy neurons. In lambs (and adult ewes) on a low level of nutrition (top right panel), a fall in leptin suppresses GnRH release by inhibiting kisspeptin via MSH; it is unclear if leptin also has direct effects on KNDy neurons. In breeding season adults (middle panels), E₂ acts during the follicular phase to decrease kisspeptin release and thus GnRH pulse amplitude and progesterone acts during the luteal phase to inhibit pulse frequency via an increase in dynorphin. In follicular phase ewes, stressors inhibit GnRH pulse frequency via cortisol, which may act to increase dynorphin output from KNDy neurons (bottom left panel) and unidentified neural inputs (not illustrated). In anoestrous ewes (bottom right panel) E₂ acts via afferent input to increase DA release from A15 neurons that innervate KNDy neurons and inhibit kisspeptin and thus GnRH pulse frequency.