2 Intrafollicular injection of asprosin in water buffaloes and the potential role of FBN1 mRNA and asprosin in follicular function

Abstract

Fibrillin-1 (FBN1) functions as a structural protein in the ovary, whereas the role of its protein product asprosin remains unknown. Both proteins are encoded by the FBN1 gene; when it is cleaved at the C-terminal end, asprosin is produced. Asprosin acts as an orexigenic hormone and is associated with various metabolic parameters and sex related hormones in women. One goal of this research was to quantify FBN1 and the presumed asprosin receptor, olfactory receptor family 4 subfamily M member 1 (OR4M1) mRNA in water buffalo granulosa cells (GC) and correlate them to aromatase (CYP19A1) gene expression. A second goal was to determine the effect of asprosin on follicular growth in vivo. In Experiment 1, ovaries were collected from a local slaughterhouse, GC from small (<6 mm) and large (6–13 mm) follicles were aspirated, RNA was extracted, and gene expression analysis conducted. In Experiment 2, an intrafollicular injection of asprosin (6 μL of asprosin in 194 μL of phosphate-buffered saline; to achieve 20 ng mL⁻¹) or vehicle (200 μL of phosphate-buffered saline; Controls) was given via the ovarian stroma below the dominant follicle of synchronized cows (n = 5/group) 1 day after injection of prostaglandin F_2α, and follicle sizes were measured daily via transrectal ultrasonography until the day of ovulation. Means were compared using t-test for gene expression analysis, and Pearson correlation coefficients calculated among FBN1, OR4M1, and CYP19A1 gene expression. A repeated-measures ANOVA was used to determine the effect of asprosin on follicle size and growth rate of follicles. In Experiment 1, FBN1 mRNA abundance was 7.51-fold greater in GC of small than large follicles (P < 0.05). There was no significant difference in the OR4M1 (57.83 ± 39.89 vs. 38.98 ± 4.86) or CYP19A1 (11.46 ± 3.72 vs. 8.27 ± 4.81) mRNA abundance between the 2 sizes of follicles (P > 0.10). Abundance of CYP19A1 mRNA was positively correlated with FBN1 (r = 0.55, P < 0.05) and OR4M1 mRNA (r = 0.50, P < 0.05). In Experiment 2, there was a treatment × day interaction (P < 0.10) for follicle size and growth rate of follicles. Cows treated with asprosin had a higher growth rate from Day 1 to 2 (1.09 ± 0.39 to 2.37 ± 0.32 mm/day) than placebo cows (1.74 ± 0.55 to 1.05 ± 0.61 mm/day) after injection. Most of the follicles from both treatment groups ovulated 3 days post injection. These findings suggest that FBN1 (and thus asprosin) are present in buffalo GC and may be developmentally expressed. Also, asprosin may induce follicular growth when given in vivo. Whether these proteins directly regulate aromatase expression, and therefore oestradiol production, during follicle development will require further study.