Sodium Fluoride In Vitro Treatment Affects the Expression of Gonadotropin and Steroid Hormone Receptors in Chicken Embryonic Gonads

Abstract

Sodium fluoride (NaF), in addition to preventing dental decay may negatively affect the body. The aim of this study was to examine the effect of a 6 h in vitro treatment of gonads isolated from 14-day-old chicken embryos with NaF at doses of 1.7 (D1), 3.5 (D2), 7.1 (D3), and 14.2 mM (D4). The mRNA expression of luteinizing hormone receptor (LHR), follicle-stimulating hormone receptor (FSHR), estrogen receptors (ESR1 and ESR2), progesterone receptor (PGR), and the immunolocalization of progesterone receptors were examined in the tissue. In the ovary, the expression of FSHR and LHR increased following the NaF treatment. In the case of FSHR the highest stimulatory effect was noticed in the D2 group, while the expression of LHR increased in a dose-dependent manner. A gradual increase in ESR1 and PGR mRNA levels was also observed in the ovary following the NaF treatment, but only up to the D3 dose of NaF. The highest ESR2 level was also found in the D3 group. In the testes, the lowest dose of NaF significantly decreased the expression of FSHR, ESR1, ESR2, and PGR. On the other hand, an increase in PGR expression was observed in the D3 group. The expression of LHR in the testes was not affected by the NaF treatment. Immunohistochemical analysis showed that NaF exposure increased progesterone receptor expression in the ovarian cortex, while it decreased its expression in the testes. These results reveal that NaF may disturb the chicken embryonic development and different mechanisms of this toxicant action exist within the females and males.

Keywords: NaF; chicken embryo; gonads; hormone receptors.

Figure 1

The mRNA expression of genes encoding gonadotropin receptors: follicle stimulating hormone receptor (FSHR) (A) and luteinizing hormone receptor (LHR) (B), in the ovary and testes of chicken embryos at the 14th day of embryogenesis, following an in vitro treatment with increasing doses of sodium fluoride (NaF). Each value represents the mean ± SEM from n = 6 (six embryonic gonads). Data of gene expression represent the mean of relative quantity (RQ), standardized to control expression in the ovary (RQ = 1). Statistical differences (p < 0.05) are marked by letters.

Figure 2

The mRNA expression of genes encoding estrogen receptors α (ESR1) (A) and β (ESR2) (B), and progesterone receptor (PGR) (C), in the ovary and testes of chicken embryos at the 14th day of embryogenesis, following an in vitro treatment with increasing doses of NaF. Each value represents the mean ± SEM from n = 6 (six embryonic gonads). Data of gene expression represent the mean of relative quantity (RQ), standardized to control expression in the ovary (RQ = 1). Statistical differences (p < 0.05) are marked by letters.

Figure 3

Immunolocalization of the progesterone receptor (PGR) in the control and NaF treated chicken embryonic ovaries. M: Ovarian medulla; C: Ovarian cortex; Er: Erythrocyte (autofluorescence, see Supplementary Materials 1 and 2). Arrows: Immunopositive reaction specific for the progesterone receptor (red fluorescence); DAPI: Blue fluorescence of cell nuclei. Scale bar = 100 µm.

Figure 4

Immunolocalization of the progesterone receptor (PGR) in the control and NaF treated chicken embryonic testes exhibiting a developed medulla, characterized by seminiferous tubules (ST) with Sertoli cells and prospermatogonia. Arrows: Immunopositive reaction specific for the progesterone receptor (red fluorescence); DAPI: Blue fluorescence of cell nuclei. Scale bar = 100 µm.