Bisphenol S Alters the Steroidome in the Preovulatory Follicle, Oviduct Fluid and Plasma in Ewes With Contrasted Metabolic Status

Abstract

Bisphenol A (BPA), a plasticizer and endocrine disruptor, has been substituted by bisphenol S (BPS), a structural analogue that had already shown adverse effects on granulosa cell steroidogenesis. The objective of this study was to assess the effect of chronic exposure to BPS, a possible endocrine disruptor, on steroid hormones in the ovary, oviduct and plasma using the ewe as a model. Given the interaction between steroidogenesis and the metabolic status, the BPS effect was tested according to two diet groups. Eighty adult ewes were allotted to restricted (R) and well-fed (WF) groups, that were further subdivided into two subgroups. Ewes were exposed to 50 µg BPS/kg/day in their diet (R50 and WF50 groups) or were unexposed controls (R0 and WF0 groups). After at least 3 months of BPS exposure, preovulatory follicular fluid, oviduct fluid and plasma were collected and steroid hormones were analyzed by gas chromatography coupled with tandem mass spectrometry (GC-MS/MS). A deleterious effect of restricted diet on the volume of oviduct fluid and numbers of pre-ovulatory follicles was observed. Exposure to BPS impaired estradiol concentrations in both follicular and oviduct fluids of well-fed ewes and progesterone, estradiol and estrone concentrations in plasma of restricted ewes. In addition, a significant interaction between metabolic status and BPS exposure was observed for seven steroids, including estradiol. In conclusion, BPS acts in ewes as an endocrine disruptor with differential actions according to metabolic status.

Keywords: bisphenol S; endocrine disruptors; ewe; metabolic status; steroidome.

Figure 1

Biosynthesis pathways of steroid hormones. All steroids are derived from cholesterol, which is first converted into pregnenolone and progestogens. Progesterone is the major steroid of the progestogen pathway. This hormone can be converted to androstenedione, then to testosterone. Testosterone is the main steroid in the androgen pathway. This steroid is a precursor to the estrogenic pathway, in which estradiol is the main steroid. The corticoid pathway is derived from the progestogen pathway, with mineralocorticoids (corticosterone) and glucocorticoids (cortisol). CYP, Cytochrome P450 Family; HSD, Hydroxysteroid dehydrogenase; SRD5A, 3-oxo-5α-steroid 4-dehydrogenase.

Figure 2

Experimental design. A total of 80 ewes were allocated to one of two diet groups (Restricted, R and Well-Fed, WF), and, in each group, in one of two exposure subgroups (0 or 50 µg BPS/kg/day). Thus, four experimental groups of ewes were formed: restricted without BPS (R0, n = 20), restricted with 50 µg/kg/d of BPS (R50, n = 20), well-fed without BPS (WF0, n = 20), and well-fed with 50 µg/kg/d of BPS (WF50, n = 20). The diet and BPS exposure lasted for at least 3 months before the start of plasma/fluid collection. Between September and December, the estrous cycle of ewes were sequentially synchronized (batches of 6-8 ewes, represented by black arrows), and animals were slaughtered 2 days after PMSG administration for collection of plasma, oviductal fluid (OF), and preovulatory follicular fluid (POFF, diameter ≥ 6 mm), and follicular fluid (FF, < 6mm follicles, n = 20, 5 ewes per experimental groups). NEFA: non-esterified fatty acids.

Figure 3

GC-MS/MS steroid hormones measurements in the preovulatory follicular fluid (POFF) of ewes according to their metabolic status and exposure to BPS. Concentrations of progestogens (A, B), corticoids (C, D), androgens (E, F) and estrogens (G, H) in pooled (3-4 ewes per pool) preovulatory follicular fluid (POFF) of ewes were assessed according to their metabolic status (restricted or well-fed) and previous dietary exposure to bisphenol S (0 or 50 µg/kg/day). Concentrations of cortisol (C) were not detectable in this biological fluid. Results are presented as mean +/- SEM (R0, n = 4; R50, n = 4; WF0, n = 6; WF50, n = 5). Bars with different letters indicate a significant difference (p < 0.05).

Figure 4

GC-MS/MS steroid hormones measurements in the oviductal fluid (OF) of ewes according to their metabolic status and exposure to BPS. Concentrations of progestogens (A, B), corticoids (C, D) androgens (E, F) and estrogens (G, H) in pooled (3-4 ewes per pool) oviduct fluid (OF) of ewes were assessed according to their metabolic status (restricted or well-fed) and previous dietary exposure to bisphenol S (0 or 50 µg/kg/day). Concentrations of corticoids (C, D) were not detectable in this biological fluid. Results are presented as mean +/- SEM (R0, n = 4; R50, n = 4; WF0, n = 6; WF50, n = 5). Bars with different letters indicate a significant difference (p < 0.05).

Figure 5

GC-MS/MS steroid hormones measurements in the plasma of ewes according to their metabolic status and exposure to BPS. Concentrations of progestogens (A, B), corticoids (C, D), androgens (E, F) and estrogens (G, H) in pooled (3-4 ewes per pool) circulating plasma of ewes were assessed according to their metabolic status (restricted or well-fed) and previous dietary exposure to bisphenol S (0 or 50 µg/kg/day). Results are presented as mean +/- SEM (R0, n = 4; R50, n = 4; WF0, n = 6; WF50, n = 5). Bars with different letters indicate a significant difference (p < 0.05).

Figure 6

ELISA steroid hormones measurements in individual preovulatory follicular fluid (POFF) of ewes according to their metabolic status and exposure to BPS. Concentrations of progesterone (A) and estradiol (B) in individual preovulatory follicular fluid (POFF) of ewes were assessed according to their metabolic status (restricted or well-fed) and dietary exposure to bisphenol S (0 or 50 µg/kg/day). Results are presented as mean +/- SEM (R0, n = 18; R50, n = 19; WF0, n = 20; WF50, n = 18). Bars with different letters indicate a significant difference (p < 0.05).