The origin of serotonin in the ovary is the key question for understanding mechanisms of serotonergic regulation of reproductive function. We performed a study of the expression and functional activity of the serotonin transporter (SERT) and the enzyme for the synthesis of serotonin, aromatic l-amino acid decarboxylase (DDC) in mouse ovary. A pronounced peak of SERT mRNA expression occurs at the age of 14 days, but serotonin synthesis enzymes are expressed at the maximum level in the ovaries of newborn mice. SERT is detected immunohistochemically in all cellular compartments of the ovary with a maximum level of immunostaining in the oocytes of growing ovarian follicles. DDC immunolocalization, in contrast, is detected to a greater extent in primordial follicle oocytes, and decreases at the later stages of folliculogenesis. Serotonin synthesis in all cellular compartments occurs at very low levels, whereas specific serotonin uptake is clearly present, leading to a significant increase in serotonin content in the oocytes of growing primary and secondary follicles. These data indicate that the main mechanism of serotonin accumulation in mouse ovary is its uptake by the specific SERT membrane transporter, which is active in the oocytes of the growing ovarian follicles.
Using RT-PCR, we showed the presence of Tph1 mRNA in follicular cells and Tph2 mRNA in oocytes isolated from primary multilayer ovarian follicles of mouse and the absence of Ddc expression, which indicates that serotonin cannot be synthesized in a developing ovarian follicle. The membrane serotonin transporter Sert is expressed in follicular cells and oocytes. Experiments on the cultivation of follicles in vitro confirmed the absence of serotonin synthesis from 5-hydroxytryptophan and the presence of membrane transport activity in the oocyte.
Serotonin (5-HT) plays an essential role in regulating female reproductive function in many animals. 5-HT accumulates in the mammalian ovary with the involvement of membrane serotonin transporter SERT and is functionally active in the oocytes of growing follicles, but shows almost no activity in follicular cells. In this study, we clarified the interplay between 5-HT membrane transport and its degradation by monoamine oxidase (MAO) in the mammalian ovary. Using pharmacologic agents and immunohistochemical staining of the cryosections of ovaries after serotonin administration in vitro, we demonstrated the activity of transport and degradation systems in ovarian follicles. The MAO inhibitor pargyline increased serotonin accumulation in the granulosa cells of growing follicles, indicating the activity of both serotonin uptake and degradation by MAO in these cells. The activity of MAO and the specificity of the membrane transport of serotonin was confirmed in primary granulosa cell culture treated with pargyline and fluoxetine. Moreover, the accumulation of serotonin is more effective in the denuded oocytes and occurs at lower concentrations than in the oocytes within the follicles. This confirms that the activity of SERT and MAO in the granulosa cells surrounding the oocytes impedes the accumulation of serotonin in the oocytes and forms a functional barrier to serotonin.
Regulation of the growth and development of female sex cells is one of the conservative functions of serotonin, but their specific mechanisms remain undisclosed yet. Molecular-genetic study of the presence of all components of the serotonergic system and dynamics of their expression in granulosa cells at different stages of folliculogenesis and in corpus luteum was carried out by reverse transcription with PCR and real-time PCR. Transcripts of enzymes tryptophan hydroxylase Tph1, aromatic amino acids decarboxylase Ddc and monoamine oxidase Maoa, membrane serotonin transporter Sert and vesicular monoamine transporter Vmat2, as well as serotonin receptors Htrlb, Htrld, Htr2a, Htr2b, Htr5b and Htr7 are detected in probes investigated. A quantitative study revealed that the expression of Vmat2, Htrlb, and Htr7 genes is greater in the early stages of folliculogenesis, whereas the expression of the Vmatl gene grows by the late stages of follicular development. The relative quantity of the mRNA of the Ddc, Maoa, Vmatl and Htrlb genes increases during luteinization, while the expression of the Vmat2 and Htr7 genes, on the contrary, decreases. The presence of expression of key components of the serotonergic system and its dynamics allow as to suggest that they are involved in serotonergic regulation of folliculogenesis, as well as luteinization.
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