Melatonin receptor mRNA expression in human granulosa cells

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Abstract

We have shown that the melatonin receptor agonist, 2-[125I] iodomelatonin, binds to high-affinity guanine nucleotide-sensitive sites on human granulosa (HG) cell membranes. In order to confirm the presence of melatonin receptors in HG cells, we have now used a reverse transcriptase-polymerase chain reaction (RT-PCR) procedure to examine receptor subtype expression. RT-PCR studies revealed the presence of the mt1 (Mel1a) melatonin receptor subtype in ten single or pooled HG cell samples which were obtained from 14 patients. In contrast, expression of MT2 ( Mel1b) mRNA was observed in only two of these HG samples. DNA sequencing of the mt1 PCR product confirmed its identity with the reported human mt1 melatonin receptor. The expression of mt1 and MT2 receptor mRNA in HG cells and the reported presence of melatonin in human follicular fluid indicate a potentially important role for this hormone in regulating human ovarian and reproductive function.

Introduction

The principal pineal hormone, melatonin, plays a role in regulating neuroendocrine function and circadian rhythmicity in mammals (Reiter, 1991, Niles, 1997). Molecular studies have characterized two mammalian melatonin receptor subtypes Mel1a and Mel1b (Reppert et al., 1994, Reppert et al., 1995) presently designated as mt1 and MT2, respectively (Dubocovich et al., 1998a). The mt1 receptor in the suprachiasmatic nucleus (SCN) or biological clock has been suggested as the mediator of the circadian effects of melatonin (Reppert et al., 1994). This role is now questioned in light of studies with mt1 knockout mice, which retain sensitivity to the phase shifting action of melatonin on rhythmic neuronal firing (Liu et al., 1997). However, mt1 receptors in the pars tuberalis are thought to be involved in modulating reproductive function in seasonal breeders such as sheep (Morgan et al., 1994, Reppert et al., 1994). The high expression of the MT2 receptor in the retina, as compared with brain (Reppert et al., 1995), suggests its involvement in mediating the effects of melatonin on retinal function, such as the modulation of dopamine release (Dubocovich, 1983). The MT2 receptor may also be involved in mediating the effects of melatonin on biological rhythmicity (Dubocovich et al., 1998b).

Melatonin has been implicated in the regulation of the hypothalamic-pituitary–gonadal axis in humans (Webb and Puig-Domingo, 1995, Reiter, 1998), but the sites and mechanisms await clarification. Nonetheless, the presence of high levels of melatonin in human preovulatory follicular fluid suggests that this hormone influences human ovarian and reproductive function (Brzezinski et al., 1987, Ronnberg et al., 1990). The foregoing is supported by our recent detection of high-affinity binding sites for the melatonin agonist, 2-[125I] iodomelatonin ([125I]Mel), on human granulosa cell membranes (Yie et al., 1995a). In order to confirm the presence of melatonin receptors in these cells, we have now utilized a reverse transcriptase-polymerase chain reaction (RT-PCR) procedure to examine melatonin receptor subtype expression in human granulosa cells.

Section snippets

Sample collection

Human granulosa (HG) cells were obtained during the winter (January–March 1997; January 1998), spring (April 1997), and summer (August 1997; July 1998) from preovulatory follicles of women undergoing oocyte retrieval in the in vitro fertilization program at Chedoke-McMaster Hospital, Hamilton, Ontario, Canada. Ovarian stimulation was performed with Lupron plus Pergonal or Fertinorm (Serono Labs, Norwell, USA) and follicular development was monitored by serum estradiol and ultrasonography. Human

Results and discussion

PCR amplification of cDNA, derived from winter, spring and summer HG samples, yielded mt1 products of the expected size (516 bp), as shown in Fig. 1, Fig. 2. The mt1 product was seen in all RT-PCR replications with RNA from these samples. In contrast, relatively weak expression of the presumptive MT2 mRNA was observed only in two (Fig. 3) of the ten samples examined. Cloning and sequencing of the mt1 product from one patient indicated a 99% identity with the previously described human mt1

Acknowledgements

This work was supported by a NSERC (Canada) Grant. We thank Yanjing Mao and Elizabeth LaRose for typing the manuscript.

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