ReviewKnocked down and out: PACAP in development, reproduction and feeding
Introduction
One of the most successful strategies to clarify the role of hormones in vivo is to disrupt the gene that encodes the hormone or its receptor. The manipulation of these genes is most useful after the structures of the gene, mRNA and protein are known and basic physiological studies, in vivo or in vitro, have determined the broad outline of the mechanism of action of the hormones. Against this background, the total elimination of a hormone from the germline or one-celled stage has yielded crucial information on the role of the hormone within the living integrated system in regard to development, survival, reproduction, energy balance, stress and many other basic functions. For example, the gene disruption (gene knockout) of receptors for FSH [7], LH [25], prolactin [33], estradiol [22], [26], progesterone [27] and insulin [1] have not only clarified the actions of the hormones, but revealed a number of new functions that were not previously known. We have taken a similar approach here to elucidate the role of pituitary adenylate cyclase-activating polypeptide (PACAP) in living organisms.
Although PACAP was discovered recently, many aspects of the hormone are known: its gene, mRNA and peptide structure, its expression in the nervous system and other tissues, and the changes that have occurred during evolution [37], [41]. In addition, three PACAP receptors have been identified by structure and include PAC1, which only binds PACAP, and receptors VPAC1 and VPAC2, which bind both PACAP and vasoactive intestinal peptide (VIP)[41], [42]. To date, a number of functions in different physiological systems have been identified for PACAP. These include roles in carbohydrate and protein metabolism [14], in the stress response [14], [16], in the regulation of other endocrine systems [41], in the immune system [41], as a neuromodulator in the sympathetic and parasympathetic nervous system [29], [35] and in the brain as a neurotrophic, neuroprotective or proliferative factor [41]. However, the question arises as to how this vast array of functions is coordinated. It is not clear whether PACAP is released in response to a basic external or internal environmental condition. One possibility is that PACAP is like prolactin, which was described as having 85 distinct effects [33]. However, the role of prolactin was substantially clarified when the prolactin receptor was knocked out and the crucial effects of prolactin on survival and reproduction were obvious, in addition to novel effects [33]. Using the same strategy, several laboratories have prepared mouse lines in which the gene was disrupted for either PACAP or PACAP receptors. In this report, we focus on the consequence of eliminating PACAP for reproduction and energy homeostasis in mice. In addition to these two basic systems, we examine early brain development and the role of PACAP, but use a more suitable model, zebrafish (Danio rerio), where rapid development occurs outside of the mother.
Section snippets
Manipulation of PACAP genes by knockdown or knockout methods
Gene knockdown is used for organisms in which gene knockout is not technically possible. Only mice among the vertebrates can be used for gene knockout to date, but the method has the advantage that the gene product is eliminated in all tissues throughout development and in adults. However, organisms other than mice are better models for specific studies. The zebrafish is excellent for developmental studies as fertilization is external and embryo development can be directly observed. Development
Zebrafish as a model
PACAP is expressed early in the brain of the mouse embryo where mRNA was detected at E9.5-10.5 and protein was measured in the rat brain at E14 [37], [41]. This evidence suggests that PACAP is present during brain development. To observe developmental changes, an alternative species to mouse, the zebrafish, was selected to avoid the influence of maternal PACAP and to use embryos that are nearly transparent for constant observation. Although PACAP is highly conserved between mammals and fish,
Background
Reproduction requires the successful completion of copulation, egg release, fertilization, embryo implantation, maintenance of pregnancy, parturition, and rearing of neonates. PACAP knockout and PAC1 receptor knockout mice both exhibited impaired fertility [38], [20], although the mechanism by which PACAP influences reproduction is only partially understood. PACAP is expressed within the hypothalamus, and has been found to regulate progesterone-mediated female receptivity in mice and rats [2].
PACAP and energy homeostasis
PACAP has a role in insulin secretion, glucose regulation [41], and lipid metabolism [14], but there has been little study to date on what, if any, role PACAP may play in general energy homeostasis. It has been suggested that PACAP inhibits feeding because intra-cerebroventricular injections of PACAP reduced short-term food intake in mice [31], but the mechanism is not known. Also, the distribution of PACAP in the regions of the hypothalamus (ventromedial nucleus and paraventricular nucleus)
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Cited by (52)
Relationships between constitutive and acute gene regulation, and physiological and behavioral responses, mediated by the neuropeptide PACAP
2022, PsychoneuroendocrinologyCitation Excerpt :The technology for gene knock-out was developed by the Capecchi lab in 1987 (Thomas and Capecchi, 1987), and originally applied to investigation of homeobox genes and their influence on mammalian development, including that of the central nervous system. Knock-out technology, applied to individual peptide and peptide receptor genes throughout the 1990′s and early 00′s (e.g., (Sherwood et al., 2007)) allowed a more definitive causality to be applied to the roles and functions of neuropeptides/neuropeptide receptors than was possible pharmacologically with chemical agonists and antagonists. However, as Hokfelt and colleagues point out, the era of molecular biology did not lead automatically to the clear identification of physiological roles for individual peptides (Hokfelt et al., 2018).
Peptidomics of the zebrafish Danio rerio: In search for neuropeptides
2017, Journal of ProteomicsPACAP system evolution and its role in melanophore function in teleost fish skin
2015, Molecular and Cellular EndocrinologyPituitary adenylate cyclase-activating polypeptide (PACAP) in fetal cord blood
2014, Early Human DevelopmentCitation Excerpt :Given its expression in placenta, further study of PACAP expression in normal and abnormal pregnancy may be informative. While its function in placenta is not known, fertility is reduced in mice lacking PACAP or PAC1-R perhaps because of impaired implantation [20]. The authors have no conflict of interest.
PACAP modulates GnRH signaling in gonadotropes
2014, Molecular and Cellular EndocrinologyCitation Excerpt :Global PACAP- and PAC1R-deficient mouse models have been generated and have suggested a role for PACAP in maintenance of normal fertility (Isaac and Sherwood, 2008; Jamen et al., 2000b; Shintani et al., 2002). It should be noted that interpretation of these models is complicated by at least two factors: (1) overlap in expression of PACAP and VIP and their receptors in reproductive tissues which may provide redundant function and, (2) widespread expression in other organ systems leading to abnormalities in thermogenesis, carbohydrate and lipid metabolism, and neurologic function which may indirectly impact reproductive capacity (Gray et al., 2001; Hashimoto et al., 2001; Jamen et al., 2000a; Sherwood et al., 2007). Jamen and colleagues have reported decreased fertility in female, but not in male, mice null for expression of the PAC1 receptor (Jamen et al., 2000b).
- 1
Present address: Diabetes Center, University of California, San Francisco, 513 Parnassus Avenue, HSW 1090 Box 0534, San Francisco, CA 94143-0534, USA.
- 2
Present address: Department of Neurology, David Geffen School of Medicine, University of California at Los Angeles, 710 Westwood Plaza, Suite C128, Los Angeles, CA 90095-1769, USA.