Serotonin Transporter Activity in Mouse Oocytes Is a Positive Indicator of Follicular Growth and Oocyte Maturity

Abstract

Serotonin (5-hydroxytryptamine, 5-HT) is known to be a regulator of oocyte maturation in a large number of animal species. In maturing mammalian oocytes, the accumulation of exogenous, maternal serotonin occurs due to the activity of the membrane transporter SERT. In this work, we investigated how SERT activity in oocytes correlates with indicators of follicular selection and oocyte maturity. An immunohistochemical study showed that the difference in the 5-HT intake activity in oocytes does not correlate with the marker of apoptosis in follicular cells, but positively correlates with markers of follicular growth, such as granulosa proliferation and follicle size. Functional analysis of oocytes at different stages of maturation showed that the expression and activity of SERT increases with oocyte maturation. An in vivo experiment on administration of the selective serotonin reuptake inhibitor fluoxetine (20 mg/kg) for 7 days showed a significant decrease in the content of serotonin in both growing GV-oocytes and ovulated mature MII-oocytes. The data obtained clearly indicate that the mechanism of specific membrane transport of serotonin normally ensures the accumulation of serotonin in maturing oocytes, and can be considered as a promising positive marker of their mature status.

Keywords: SERT; SSRI; apoptosis; fluoxetine; oocyte maturation; ovary; proliferation; serotonin.

Figure 1

Correlation analysis of the intensity of serotonin accumulation by oocytes with morphological and functional indicators of the quality of ovarian follicles. (a,c,e) Cryosections of ovarian tissue of 14 dpp females incubated with 1 μM serotonin for 2 h, co-immunostained with antibodies against serotonin and an apoptosis marker—the active form of caspase 3 (a), a proliferation marker of Ki67 (b), and an LCA-FITC dye that reveals the boundaries of the follicles (e). Scale bar 100 µm. (b,d,f) Correlation matrices reflecting the interdependence of the degree of accumulation of serotonin by oocytes with the level of apoptosis (b) and proliferation (d) in follicular cells, as well as with the size of the follicle (f).

Figure 2

Serotonin uptake activity in mouse oocytes of different maturation stages. (a) Immunohistochemical detection of serotonin in oocytes at different stages of maturation after 2 h of incubation with 1 µM serotonin. Nuclear staining with DAPI was used to reveal the structure of chromatin in GV-oocytes: non-surrounded nucleolus (NSN) and surrounded nucleolus (SN). Scale bar 50 µm. (b) Quantification of anti-serotonin immunoreactivity in oocytes at different stages of maturation after 2 h of incubation with 1 µM serotonin. Asterisk indicates significant differences between groups using Kruskal–Wallis test with Dunn’s multiple comparisons test, * p < 0.05. (c) Western blot analysis of the content of immature (70 kDa) and glycosylated mature (100 kDa) forms of SERT in GV- and MII-oocytes. (d) Western blot data quantification. RPL—relative protein level normalized to β-tubulin protein content.

Figure 3

Effect of 7-day administration of fluoxetine (20 mg/kg) on serotonin content in oocytes. (a–d) Immunohistochemical detection of serotonin on ovarian tissue cryosections (a,c) and in ovulated MII-oocytes (b,d) of control (NaCl) (a,b) and experimental (FLU) (c,d) females. Scale bar 50 µm. (e) Quantification of anti-serotonin immunoreactivity in GV- and MII-oocytes of control (Ctrl) and experimental (FLU) females. Asterisks indicate significant differences between NaCl and FLU groups using Mann–Whitney test, * p < 0.01, ** p < 0.0001. (f) HPLC analysis of the serotonin content in the blood serum of control (NaCl) and experimental (FLU) females. Asterisks indicate significant differences between groups using Mann–Whitney test, ** p < 0.005.