74 ANALYSIS OF STEROID HORMONES IN BOVINE OVIDUCTAL FLUID BY GAS CHROMATOGRAPHY COUPLED WITH TANDEM MASS SPECTROMETRY

Abstract

Steroid hormones play key roles in the regulation of physiological changes in the mammalian genital tract, including the oviduct. The aim of this study was to determine the variations in steroid hormone concentrations in bovine oviducal fluid (OF) during the oestrous cycle. Bovine oviducts were collected at a local slaughterhouse and classified into 4 stages according to the ovarian and corpus luteum (CL) morphologies (n = 18–25 cows/stage): post-ovulatory (postov), mid luteal (mid-lut), late luteal (late-lut), and preovulatory (preov). Follicular fluid was also collected from the preov follicles. Animals with follicular oestradiol-17β (E2) <40 ng mL^–1 or progesterone (P4) >160 ng mL^–1 (cystic follicles) or E2 : P4 ratio <1 (atretic follicles) were excluded. Oviducal fluids were collected from contra- and ipsilateral (to the CL) ampullas by squeezing. They were then centrifuged and stored at –80°C. Steroids were extracted from pools of 150 to 200 µL of OF (4–10 cows/pool; 3–4 pools per “stage × side” group), purified, fractioned by high-performance liquid chromatography, derivatized, and analysed by gas chromatography coupled with tandem mass spectrometry (GC-MS/MS). Comparisons between groups were made with a two-way ANOVA followed by Bonferroni post-tests. Data are expressed as means ± SEM. A P-value < 0.05 was considered as significant. In ipsilateral OF, the concentrations of P4 increased from postov (56.9 ± 13.4 ng mL^–1) to mid-lut (120.3 ± 34.3 ng mL^–1; P < 0.01) then decreased from late-lut (76.7 ± 1.8 ng mL^–1) to preov (6.3 ± 1.7 ng mL^–1; P < 0.001) stages, and were 4 to 16 times more concentrated than in contralateral OF. Several P4 reduced metabolites (5α-dihydroP4, 20α-dihydroP4, 17α-hydroxyP4, 3α5α20α-hexahydroP4, 3β5α20α-hexahydroP4) followed the same pattern of variation. Concentrations of the P4 precursor pregnenolone were highest at postov (4.2 ± 0.3 ng mL^–1 in ipsilateral OF) then decreased from mid-lut to preov (3.4 ± 0.6 to 1.3 ± 0.1 ng mL^–1; P < 0.001) and were higher than in contralateral OF at postov (P < 0.001) and mid-lut (P < 0.01) stages. Concentrations of E2 were higher in preov (290.5 ± 63.2 pg mL^–1 in ipsilateral OF) compared to all other stages (P < 0.05) with no difference between ipsi- and contralateral sides at any stage. Concentrations of the oestrogen precursors androstenedione and testosterone displayed the same pattern of variation as E2. In contrast, concentrations of dehydroepiandrosterone and estrone did not vary between stages or sides relative to the CL. In addition, high concentrations of cortisol and cortisone were found in ipsi- and contralateral OF at all stages of the oestrous cycle (on average 48.6 and 31.9 ng mL^–1, respectively). In conclusion, numerous steroids, steroid precursors, and metabolites were found in the bovine OF. The local transfer of ovarian steroids creates a highly concentrated and fluctuating hormonal environment in the ipsilateral OF during the oestrous cycle. These results could be useful to improve media for IVF, development, and oviducal cell culture.