Serotonin and Its Antidepressant-Sensitive Transport in Mouse Cumulus-Oocyte Complexes and Early Embryos1

Abstract

Serotonin (5-hydroxytryptamine [5-HT]), is a neurohormone found in various nonneural tissues, including the gonads of many invertebrates, in which it regulates spawning and oocyte meiotic maturation. The possibility that a local serotonergic network might also exist in the female gonads of vertebrate species, including mammals, remains poorly documented. To clarify this possibility, we investigated mouse cumulus cells, oocytes, and embryos for three key serotonergic components, namely, 5-HT itself; the rate-limiting enzyme for its production, tryptophan hydroxylase 1 (TPH1); and the 5-HT-specific transporter (SLC6A4) required for modulating its cellular effects. Using a combination of reverse transcription-polymerase chain reaction analysis and immunofluorescence confocal microscopy, we showed that mouse cumulus cells, oocytes, and embryos contain 5-HT and SLC6A4, while only cumulus cells possess the 5-HT-producing enzyme TPH1 and may thus be the local source of 5-HT observed in their neighboring cells. With a semiquantitative assay in single cells, we demonstrated that 5-HT can actively be taken up by isolated oocytes when it is supplied exogenously in vitro. This 5-HT transport in isolated oocytes is driven by a classical serotonin transporter, expressed up to the blastocyst stage, that is sensitive to the antidepressants fluoxetine and fluvoxamine, which belong to the selective serotonin reuptake inhibitor family. All together, our results show that 5-HT may be produced locally by cumulus cells and that it can be actively taken up by mammalian oocytes and embryos as part of a likely larger serotonergic network possibly regulating various developmental processes much earlier than previously thought.

Serotonin in pre-implantation mouse embryos is localized to the mitochondria and can modulate mitochondrial potential

“…This is similar to two previous reports where similar puncta were observed using anti-serotonin antibodies. However, both studies showed puncta with diffuse staining that could have been the result of fixation (Il'kova et al 2004, Amireault & Dube 2005. The fluorescent puncta we observed were not only diffuse but also did not have a vesicular shape.…”

“…The antibody used in this particular study did not distinguish between TPH1 and TPH2 proteins. Another study failed to detect Tph1 transcripts in embryos and oocytes (Amireault & Dube 2005). This study did not, however, examine for Tph2 transcripts.…”

“…We reasoned that, being a charged molecule, large concentrations of serotonin within or associated with mitochondria might affect its potential. Since incubation with serotonin is known to increase serotonin-specific staining in mouse pre-implantation embryos as a result of uptake (Amireault & Dube 2005), we used extracellular Figure 4 Confocal images of serotonin detected in mouse embryos by immunofluorescence staining using anti-serotonin antibody. Each image is an optical plane of (A) the oocyte, (B) zygote, (C) two-cell embryo, (D) eight-cell stage, and (E) the blastocyst.…”

“…Two recent papers have also reported the presence of serotonin in the early mouse embryo using anti-serotonin antibodies (Il'kova et al 2004, Amireault & Dube 2005. These studies reported the distribution of serotonin within fixed embryos.…”

“…This molecule is the target for a variety of clinically used antidepressants, such as fluoxetine and stimulant drugs/drugs of abuse, such as amphetamines (Scholze et al 2000, Vaswani et al 2003, Hilber et al 2005. The presence of SLC6A4 has also been reported in early embryos, suggesting that embryos may take up serotonin from the surrounding fluid (Amireault & Dube 2005). We, therefore, also examined for the presence of Slc6a4 transcripts in the various pre-implantation stages of mouse embryos.…”