Conditional Deletion of KOR in Kisspeptin Cells Does Not Alter LH Pulses, Puberty, or Fertility in Mice

Classic pharmacological studies suggested that endogenous dynorphin-KOR signaling is important for reproductive neuroendocrine regulation. With the seminal discovery of an interconnected network of hypothalamic arcuate neurons co-expressing kisspeptin, neurokinin B, and dynorphin (KNDy neurons), the KNDy hypothesis was developed to explain how gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) pulses are generated. Key to this hypothesis is dynorphin released from KNDy neurons acting in a paracrine manner on other KNDy neurons via kappa opioid receptor (KOR) signaling to terminate neural "pulse" events. While in vitro evidence supports this aspect of the KNDy hypothesis, a direct in vivo test of the necessity of KOR signaling in kisspeptin neurons for proper LH secretion has been lacking. We therefore conditionally knocked out KOR selectively from kisspeptin neurons of male and female mice and tested numerous reproductive measures, including in vivo LH pulse secretion. Surprisingly, despite validating successful knockout of KOR in kisspeptin neurons, we found no signifcant effect of kisspeptin cell-specific deletion of KOR on any measure of puberty, LH pulse parameters, LH surges, folliclestimulating hormone (FSH) levels, estrous cycles, or fertility. These outcomes suggest that the KNDy hypothesis, while sufficient normally, may not be the only neural mechanism for sculpting GnRH and LH pulses, supported by recent findings in humans and mice. Thus, besides normally acting via KOR in KNDy neurons, endogenous dynorphin and other opioids may, under some conditions, regulate LH and FSH secretion via KOR in non-kisspeptin cells or perhaps via non-KOR pathways. The current models for GnRH and LH pulse generation should be expanded to consider such alternate mechanisms. Key Words: kisspeptin, Kiss1, GnRH, KNDy, dynorphin, opioid Abbreviations: ARC, arcuate; CON, [Kiss.sup.Cre-]/Oprk[1.sup.fl/fl] mice; [E.sub.2], estradiol; FE, first estrus; FSH, follicle-stimulating hormone; GnRH, gonadotropin-releasing hormone; Kiss KORKO, [Kiss.sup.Cre+]/Oprk[1.sup.fl/fl] mice; KNDy neurons, kisspeptin/neurokinin B/dynorphin neurons; KOR, kappa opioid receptor; LH, luteinizing hormone; MOR, mu opioid receptor; NKB, neurokinin B; NK3R, neurokinin-3 receptor; OVX, ovariectomy; PND, postnatal day; PS, preputial separation; RP3V, rostral periventricular nucleus of the third ventricle; , testosterone; VO, vaginal opening.