Kisspeptin and RFamide-related peptide 3 neurons in bovine hypothalamus: estrogen receptor α expression and inputs to gonadotrophin releasing hormone neurons

Fertility in dairy cattle is considered low, most likely due to selection for high milk production resulting in extreme metabolic demands and a period of negative energy status. In most mammals, there appears to be an inverse relationship between metabolic status and reproduction. To address and manage this relationship in dairy cows, a better understanding of brain control of reproduction is required. Final output signal of the brain is gonadotropin releasing hormone (GnRH), but it is evident that integration of internal and external signals, such as feed intake and metabolic status, occurs upstream of the GnRH neurons. The RF-amide neuropeptides, kisspeptin (Kp), and RFamide-related peptide 3 (RFRP-3) may be involved in this integration and relaying this to the GnRH neurons. In cattle, GnRH secretion is stimulated by Kp and inhibited by RFRP-3. We used immunohistochemistry (IHC) to map the distributions of Kp and RFRP-3 neurons in the hypothalamus of dairy cattle, determined their coexpression with estrogen receptor alpha (ERα), and whether they made contact with GnRH neurons. The heads of male and female calves (n = 3 each), steers, heifers, and lactating cows (n = 2/group) were perfusion-fixed (4% paraformaldehyde) and the brains dissected out and frozen. Dual-label IHC for Kp or RFRP-3 with ERα or GnRH was performed on 40 micron thick cryostat sections, using fluorescently labelled secondary antibodies. GnRH neurons were identified in the preoptic area and anterior hypothalamic area. Highest numbers of neurons expressing ERα were in the arcuate nucleus, ventromedial nucleus, preoptic area, and anterior hypothalamic area. Kp neurons were located primarily in arcuate nucleus, with some cells in the preoptic area (although few were observed in calves). Most Kp neurons in adult animals of both sexes, but few in calves, expressed ERα. Very few GnRH neurons, however, received close appositions from Kp neuron fibers. RFRP-3 neurons were localised in the dorsomedial hypothalamus and paraventricular nucleus, with fewer cells observed in cows than heifers and steers, whereas ERα was not expressed in these regions. Fewer than 20% of GnRH neurons received close contact from RFRP-3 neurons. In conclusion, we inferred that feedback actions of estrogen on GnRH secretion may, in part, be relayed via Kp neurons but unlike most other mammalian species studied, this is unlikely to be a direct action. RFRP-3 neurons likely have a small role in the regulation of GnRH neurons in cattle. Further studies are required to clarify the physiological roles of Kp and RFRP-3 in regulation of GnRH secretion in dairy cattle.

Keywords: Kisspeptin, dairy cattle, GnRH, hypothalamus, neuroendocrine signalling

This manuscript was originally published in the journal Clinical Theriogenology Vol 12(3) Sept 2020.  Clinical Theriogenology is the official journal of the Society for Theriogenology (SFT) and the American College of Theriogenologists (ACT).  This content has been reproduced on the IVIS website with the explicit permission of the SFT/ACT.