Follicular fluid steroid profile in sows: relationship to follicle size and oocyte quality†

Abstract

Identification of reliable characteristics of follicle quality and developmental competence has been pursued in numerous studies, but with inconsistent outcomes. Here, we aimed to identify these characteristics by analysis of the follicular fluid (FF) steroid profile in relation to cumulus-oocyte complex (COC) morphology and follicle size, followed by molecular substantiation. Multiparous sows at weaning were used to facilitate analysis at the start of the follicular phase of the oestrus cycle. Sows with a higher average follicle size (≥5 mm vs. < 5 mm) had a higher follicular fluid β-estradiol concentration, but did not differ in other measured steroids. Sows with high compared to low percentage high-quality COCs (<70% vs. ≥70% high-quality) had follicular fluid with a higher concentration of β-estradiol, 19-norandrostenedione, progesterone, and α-testosterone, while the concentration of cortisol was lower. Transcriptome analysis of granulosa cells of healthy follicles of sows with a high percentage high-quality COCs showed higher abundance of transcripts involved in ovarian steroidogenesis (e.g., CYP19A2 and 3, POR, VEGFA) and growth (IGF1) and differential abundance of transcripts involved in granulosa cell apoptosis (e.g., GADD45A, INHBB). Differences in aromatase transcript abundance (CYP19A1, 2 and 3) were confirmed at the protein level. In addition, sows with a high percentage high-quality COCs lost less weight during lactation and had higher plasma IGF1 concentration at weaning, which may have affected COC quality. To the best of our knowledge, this study is also the first to report the relation between FF steroid profile and COC quality.

Keywords: follicle; granulosa cells; insulin-like growth factor; metabolism; porcine; steroid hormones.

Figure 1

Experimental set-up of a total of 29 multiparous sows; left ovaries were used for follicle aspiration of the 15 largest follicles. Follicular fluid was used for steroid profiling using a modified UHPLC-MS/MS method, and cumulus-oocyte complexes (COCs) were morphologically classified as high-quality or low-quality to determine the percentage of high-quality COCs for each sow. Sows with < 70% high-quality COCs were classified as low-COC-quality and sows with ≥70% high-quality COCs as high-COC-quality. Right ovaries were used to determine follicle size and health status of all visible follicles of the right ovary, by using a cleaved-caspase 3 staining. Average follicle size of the 15 largest follicles was used to determine the follicle size class (small-FS < 5 mm vs. large-FS ≥ 5 mm). A subset of 8 out of 29 sows was used for whole-genome transcriptome analysis of granulosa cells, of which 4 were classified as low-COC-quality (54 ± 12% high-quality COCs) and 4 were classified as high-COC-quality (93 ± 15% high-quality COCs), as based on the left ovary. For each sow, granulosa cells of 6 healthy follicles of the pool of 10 largest healthy follicles were isolated using laser capture microdissection, after which samples were individually hybridized onto whole-genome microarrays for whole- genome transcriptome analysis.

Figure 2

Follicular fluid concentrations of progesterone (A), 19-norandrostenedione (B), α-testosterone (C), β-estradiol (D), and cortisol (E) ( ng/ml) of the 15 largest follicles of the left ovary of sows with high-COC-quality (≥70% high-quality COCs, N = 14) vs. sows with low-COC-quality (<70% high-quality COCs, N = 15). ^*P-value < 0.05.

Figure 3

Follicular fluid steroid concentrations (ng/ml) of sows with a small average follicle size (small-FS: average follicle size of the 15 largest follicles of the right ovary ≤ 5 mm, N = 14) vs. sows with a large follicle size (large-FS: average follicle size ≥5 mm, N = 15). ^*P-value < 0.05.

Figure 4

Aromatase protein abundance in granulosa cells of healthy follicles of sows with high-COC-quality (98 ± 5% high-quality COCs (N = 6)) vs. low-COC-quality (37 ± 19% high-quality COCs (N = 6)) in the smallest and largest follicle of the 10 largest healthy follicles. Data are presented as average ± SD. ^*P-value < 0.05.