Fast free of acrylamide clearing tissue (FACT) for clearing, immunolabelling and three-dimensional imaging of partridge tissues

Fast free of acrylamide clearing tissue (FACT) is a modified sodium dodecyl sulfate-based clearing protocol for the chemical clearing of lipids that completely preserves fluorescent signals in the cleared tissues. The FACT protocol was optimized to image translucent immunostained brain and non-nervous tissues. For this purpose adult male Chukar partridge (Alectoris chukar) was used as a model. After clearing the tissues, 1 or 2 mm-thickness sections of tissues were immunolabeled. The paraventricular nucleus in the hypothalamus (2-mm section) was cleared with FACT, and then was stained with gonadotropin-inhibitory hormone (GnIH) antibody and Hoechst. Simultaneously, immunohistochemical (IHC) staining of cryosectioned brain (30 μm) was done by GnIH-antibody. The FACT protocol and staining of cell nuclei of nine other tissues were done by a z-stack motorized fluorescent microscope. GnIH-immunoreactive neurons were found by FACT and IHC during the breeding season in male partridges. Deep imaging of the kidney, duodenum, jejunum, lung, pancreas, esophagus, skeletal muscle, trachea, and testis were also done. The FACT protocol can be used for the three-dimensional imaging of various tissues and immunostained evaluation of protein markers

The role of neuropeptides and neurotransmitters on kisspeptin/kiss1r-signaling in female reproduction

Reproductive function is regulated by the hypothalamic-pituitary-gonads (HPG) axis. Hypothalamic neurons synthesizing kisspeptin play a fundamental role in the central regulation of the timing of puberty onset and reproduction in mammals. Kisspeptin is a regulator of gonadotropin releasing hormone (GnRH) and luteinizing hormone (LH). In female rodent, the kisspeptin (encoded by kiss1 gene), neurokinin B (Tac3) and dynorphin neurons form the basis for the “KNDy neurons” in the arcuate nucleus and play a fundamental role in the regulation of GnRH/LH release. Furthermore, various factors including neurotransmitters and neuropeptides may cooperate with kisspeptin signaling to modulate GnRH function. Many neuropeptides including proopiomelanocortin, neuropeptide Y, agouti-related protein, and other neuropeptides, as well as neurotransmitters, dopamine, norepinephrine and γ-aminobutyric acid are suggested to control feeding and HPG axis, the underlying mechanisms are not well known. Nonetheless, to date, information about the neurochemical factors of kisspeptin neurons remains incomplete in rodent. This review is intended to provide an overview of KNDy neurons; major neuropeptides and neurotransmitters interfere in kisspeptin signaling to modulate GnRH function for regulation of puberty onset and reproduction, with a focus on the female roden