Immunohistochemical characterization of the arcuate kisspeptin/neurokinin B/dynorphin (KNDy) and preoptic kisspeptin neuronal populations in the hypothalamus during the estrous cycle in heifers

Abstract

Elucidating the physiological mechanisms that control reproduction is an obvious strategy for improving the fertility of cattle and developing new agents to control reproductive functions. The present study aimed to identify kisspeptin neurons in the bovine hypothalamus, clarifying that a central mechanism is also present in the cattle brain, as kisspeptin is known to play an important role in the stimulation of gonadotropin-releasing hormone (GnRH)/gonadotropin secretion in other mammals. To characterize kisspeptin neurons in the bovine hypothalamus, the co-localizations of kisspeptin and neurokinin B (NKB) or kisspeptin and dynorphin A (Dyn) were examined. Hypothalamic tissue was collected from Japanese Black or Japanese Black × Holstein crossbred cows during the follicular and luteal phases. Brain sections, including the arcuate nucleus (ARC) and the preoptic area (POA), were dual immunostained with kisspeptin and either NKB or Dyn. In the ARC, both NKB and Dyn were co-localized in kisspeptin neurons during both the follicular and luteal phases, demonstrating the presence of kisspeptin/NKB/Dyn-containing neurons, referred to as KNDy neurons, in cows. In the POA, no co-localization of kisspeptin with either NKB or Dyn was detected. Kisspeptin expression in the follicular phase was higher than that in the luteal phase, suggesting that kisspeptin expression in the POA is positively controlled by estrogen in cows. The kisspeptin neuronal populations in the ARC and POA likely play important roles in regulating the GnRH pulse and surge, respectively, in cows.

Fig. 1.

Plasma estradiol (A) and progesterone (B) concentrations during the follicular (n = 4) and luteal (n = 4) phases in cows. Values are shown as mean ± SEM. * P < 0.05 between the follicular and luteal phases.

Fig. 2.

Expression of kisspeptin, neurokinin B (NKB), and dynorphin A (Dyn) in the hypothalamic arcuate nucleus (ARC) in cows. (A) Representative photomicrographs showing dual immunohistochemistry for kisspeptin (red) and NKB (green) during the follicular and luteal phases. The merged images show the co-localization of kisspeptin and NKB in the ARC. (B) Representative photomicrographs showing dual immunohistochemistry for kisspeptin (red) and Dyn (green) during the follicular and luteal phases. The merged images show the co-localization of kisspeptin and Dyn in the ARC. The white boxes in the merged images show the areas of the magnified images. Scale bars are 100 μm and 10 μm in the merged and magnified images, respectively. 3V, third ventricle.

Fig. 3.

Expression of kisspeptin, neurokinin B (NKB), and dynorphin A (Dyn) in the preoptic area (POA) in cows. (A) Representative photomicrographs showing dual immunohistochemistry for kisspeptin (red) and NKB (green) during the follicular and luteal phases. The merged images show the absence of co-localization of kisspeptin and NKB in the POA. (B) Representative photomicrographs showing dual immunohistochemistry for kisspeptin (red) and Dyn (green) during the follicular and luteal phases. The merged images show the absence of co-localization of kisspeptin and Dyn in the POA. The white boxes in the merged images show the areas of the magnified images. Scale bars are 100 μm and 10 μm in the merged and magnified images, respectively. 3V, third ventricle.