Phenotypic characterization of reproduction in female mice lacking pituitary adenylate cyclase-activating polypeptide (PACAP)




Isaac, Emma

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Pituitary adenylate cyclase-activating polypeptide (PACAP) is a ubiquitous peptide hormone involved in the regulation of several physiological processes. This hormone is a member of the glucagon superfamily of hormones, which are primarily involved in regulation of metabolism and growth. PACAP shows the highest sequence conservation of this superfamily during evolution, suggesting a critical role for PACAP in species survival. Mice lacking PACAP (Adcyap 1) show high mortality during the postnatal period, as well as impaired reproduction in females. In this thesis I have characterized the reproductive phenotype in female mice lacking PACAP due to disruption (knockout) of the single copy pacap gene (Adcyapl ). Previous experiments in other laboratories have described reduced fertility in mice lacking PACAP or one of its receptors, the PAC] receptor. However, the mechanism by which PACAP acts to enhance reproduction has not yet been elucidated. The purpose of this study was to determine the site(s) of action of PACAP in the cascade of reproductive events from puberty to implantation. I hypothesized that PACAP was an important regulator of an early phase of reproduction, and that lack of PACAP would result in impaired reproductive success. Initially I examined puberty onset, estrous cycles, mating behaviour, and delivery of live offspring. Estrous cycles were evaluated through vaginal cytology, both in juvenile mice, to confirm that puberty had occurred, and in adult females, to determine if they maintained a normal 4-5 day estrous cycle. Mating behaviour was evaluated through a natural mating strategy, with daily assessment of the presence of a copulatory plug. The reproductive outcome of delivering live pups was characterized, and pup survival was followed. I then characterized PACAP null mice as to ovulation, ovarian histology and fertilization of eggs in vivo. Finally, I measured implantation rates in PACAP null females, as well as levels of the hormone prolactin, an essential regulator of early pregnancy. In the present study I found a number of reproductive functions that were normal without PACAP. Puberty onset was unaffected in knockout mice and they displayed regular 4-5 day estrous cycles. Also. PACAP null females mated when paired with a male of proven fertility. However, mating behaviour follows an unusual pattern in PACAP null mice, where 33% mate on more than one day during a week-long pairing unlike wild-type mice that would normally mate only once. Also, significantly fewer PACAP null females than wild-type females gave birth following mating: 21% and 100%, respectively. Ovulation and ovarian histology were normal in PACAP null females, as was fertilization of released eggs. However, only 13% of PACAP null females had implanted embryos 6.5 days after mating. The mechanism of impaired implantation may be a defect in prolactin secretion. Prolactin levels were significantly lower in PACAP null females than in wild-types following mating. Prolactin is an essential hormone for the support of early pregnancy. These results support the conclusion that PACAP acts as an important regulator of prolactin in reproduction. It is possible that PACAP in the hypothalamus is the dominant prolactin-releasing factor, which has been sought many years but never definitively identified.



peptide hormones, mice, reproduction