Validation of a sensitive direct assay for melatonin for investigation of circadian rhythms in different species

Abstract

The role of melatonin in animals which do not show marked seasonal changes in reproduction is disputed, in part because of the wide variation in reported concentrations. One reason for this may be the difficulties associated with the measurements of low molar concentrations of melatonin and the presence of a wide variety of potentially cross-reacting substances. The availability of a high affinity antiserum has allowed an assay, with low cross-reactivity and good sensitivity, to be established for the direct measurement of melatonin in a wide range of biological fluids, in particular serum, plasma and follicular fluid from man and rat. The high affinity of the antiserum enabled a tritium label of high specific activity to be used, removing the problems associated with the iodination of a small molecular weight compound. Melatonin concentrations in the assay were evaluated by four different methods: UV absorbance, gas chromatography, comparison of the immunoreactive concentrations of the label with the expected concentration by dilution and by comparison with a previously established assay which uses the same antiserum. Melatonin was measured in serum from twelve healthy women over two 24-h periods; eight women with normal menstrual cycles and four taking the contraceptive pill. Concentrations were found to range from 19.8 to 215 pmol/l during the day in both groups. In women with normal menstrual cycles peak concentrations of 513.2 +/- 54.1 (S.E.M.) pmol/l were recorded at 04.00 h, whereas higher concentrations were found in women taking the pill, reaching a peak of 849.12 +/- 21.8 (S.E.M.) pmol/l at 04.00 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Melatonin deprival modifies follicular and corpus luteal growth dynamics in a sheep model

“…These findings are confirmed by results of this study, showing that both P 4 production and corpus luteum growth were impaired in pinealectomised ewes. This result may be explained taking into account that melatonin remarkably increases mRNA expression of the LHCGR (but not FSHR) in human granulosa-luteal cells (Woo et al 2001).…”

“…A previous study reported that pinealectomised ewes can have a normal pregnancy and deliver a healthy newborn (Kennaway et al 1985). More recent studies suggest that melatonin have a role in the process of ovulation (Tamura et al 2009), considering the presence of both types of membrane melatonin receptors in granulosa cells and that melatonin can up-regulate LHCGR mRNA (Woo et al 2001). The LH is essential for the initiation of luteinisation.…”

“…Melatonin-binding sites have been detected in granulosa-luteal cells in humans (Yie et al 1995, Woo et al 2001) and in cattle (Wang et al 2012). Previous studies demonstrated that melatonin significantly increased P 4 production in in vitro-cultured human and bovine granulosa cells (Webley & Luck 1986).…”

“…In short, melatonin was assayed by a direct RIA method with 2-[125I]iodomelatonin as a tracer (Vakkuri et al 1984) and a previously described anti-melatonin antiserum (Tillet et al 1986) (melatonin Fluka-63610). This direct assay, originally described by Fraser et al (1983), was performed according to the modifications of Webley et al (1985) and Ravault et al (1989). Briefly, 100 ml rabbit anti-melatonin antiserum (final dilution: 1/400 000) and 300 ml 2-[125I]iodomelatonin (15 000 c.p.m./tube) were added to 100 ml assay buffer (tricine) or undiluted plasma.…”

“…However, the presence of high melatonin levels in follicular fluid (Brzezinski et al 1987) and the presence of receptors in granulosa cells (Yie et al 1995, Niles et al 1999, Woo et al 2001, Soares et al 2003a, Wang et al 2012, Barros et al 2013 have suggested a role of melatonin on ovarian function. This hypothesis has been confirmed by recent studies reporting strong evidence for melatonin action on the ovary, such as modulation of ovarian steroidogenesis (mainly progesterone (P 4 ) production; Taketani et al 2011) and contribution in the maintenance of a proper follicular structure and function (Soares et al 2003a,b, Barros et al 2013, Maganhin et al 2013.…”