MON-012 The Direct Effect of Kisspeptin on Human Ovarian Granulosa Cells to Regulate Steroidogenesis

Kisspeptin has a central role to stimulate the hypothalamic-pituitary-gonadal (HPG) axis. Furthermore, a previous study has suggested that kisspeptin might have a peripheral role in follicular development (1). This study aimed to 1) explore the effect of kisspeptin on CYP19A1 (aromatase) mRNA expression in human granulosa cells and aromatase concentrations in the supernatant; and 2) investigate the effect of kisspeptin on FSHR mRNA expression in human granulosa cells. In this study, human granulosa-like tumor cell line (KGN) (n=3) was incubated for 24 hours with FSH (10-8 M); FSH with IGF-1 (10-8 M); different doses of kisspeptin including 1, 10, 100, 1,000, and 10,000 nM; FSH with different doses of kisspeptin; and FSH with IGF-1 together with different doses of kisspeptin. FSH treatment alone or FSH with IGF-1 did not increase CYP19A1 mRNA expression when compared to control. Interestingly, kisspeptin treatment at the doses of 100 nM (P=0.028), 1,000 nM (P=0.005), and 10,000 nM (P=0.009) in the presence of FSH together with IGF-1 enhanced CYP19A1 mRNA expression when compared with control. Furthermore, FSH or FSH with IGF-1 or FSH with all doses of kisspeptin or FSH with IGF-1 together with all doses of kisspeptin increased aromatase concentrations in the supernatant when compared to control (P<0.01 all). Surprisingly, kisspeptin at the dose of 10,000 nM with FSH or FSH together with IGF-1 statistically increased aromatase concentrations in the supernatant when compared with FSH treatment alone or FSH with IGF-1 treatment (P<0.01 all). FSHR mRNA expression was comparable between control and all treatments. As a result, kisspeptin combined with FSH and IGF-1 could enhance CYP19A1 mRNA expression in human granulosa cells and the high dose of kisspeptin (10,000 nM) might be able to augment aromatase secretion in the supernatant. These results suggest that kisspeptin might enhance aromatase expression and secretion, which probably leads to enhance estrogen synthesis. Further studies regarding kisspeptin treatment on estrogen synthesis or secretion in human granulosa cells should be confirmed. Reference: (1) Fernandois D, et al. J Endocrinol. 2016;228(3):161-70.

FLUOXETINE MODULATES SEX STEROID LEVELS IN VITRO

Background and aims. Selective serotonin reuptake inhibitors (SSRIs) are antidepressants increasingly prescribed against depression during and after pregnancy. However, these compounds cross the placenta and are found in breast milk, thus reaching, and possibly affecting, the fetus and infant during critical developmental stages. Fluoxetine (FLX), a widely used SSRI, can interfere with estrogen signaling, which is important for the development of female sex organs and certain brain areas, among others. Interference with estrogen signaling can take place on different levels, e.g., by affecting receptor activity or hormone levels. FLX has previously been shown to induce estrogen receptor-dependent transcription in vitro at high concentrations. In this study we set out to assess effects of FLX on estradiol levels in vitro.Methods. FLX was tested using the OECD recommended H295R model, a human adrenocortical carcinoma cell line that is able to produce all steroid hormones found in the gonads and adrenal glands, including estradiol and testosterone. H295R cells were incubated with different doses of FLX for 48h. Subsequently, concentrations of these two steroids were measured in cell culture medium after FLX exposure, using liquid chromatography coupled with tandem mass spectrometry. Aromatase mRNA expression was assessed using qPCR.Results. Fluoxetine significantly increased estradiol secretion in H295R cells after a 48h exposure at low, submicromolar concentrations, but showed no effects on testosterone levels or aromatase mRNA expression.Conclusion. Fluoxetine has the potential to interfere with estrogenic signaling by increasing estradiol secretion at low concentrations, which are relevant for fetal and adult human exposure.

Aromatase Expression in the Hippocampus of AD Patients and 5xFAD Mice

Numerous studies show that 17β-estradiol (E2) protects against Alzheimer’s disease (AD) induced neurodegeneration. The E2-synthesizing enzyme aromatase is expressed in healthy hippocampi, but although the hippocampus is severely affected in AD, little is known about the expression of hippocampal aromatase in AD. To better understand the role of hippocampal aromatase in AD, we studied its expression in postmortem material from patients with AD and in a mouse model for AD (5xFAD mice). In human hippocampi, aromatase-immunoreactivity was observed in the vast majority of principal neurons and signal quantification revealed higher expression of aromatase protein in AD patients compared to age- and sex-matched controls. The tissue-specific first exons of aromatase I.f, PII, I.3, and I.6 were detected in hippocampi of controls and AD patients by RT-PCR. In contrast, 3-month-old, female 5xFAD mice showed lower expression of aromatase mRNA and protein (measured by qRT-PCR and semiquantitative immunohistochemistry) than WT controls; no such differences were observed in male mice. Our findings stress the importance of hippocampal aromatase expression in neurodegenerative diseases.