Fig 1: SMAD2 overexpression and knockdown affected the secretion of gonadotropins in pituitary cells. (A–D) mRNA and protein expression of SMAD2 in pituitary cells in each group. (E) FSH and LH secretion was detected by ELISA. (F,G) mRNA and protein expression of FSHβ and LHβ in pituitary cells in each group. All data are shown as the mean ± SEM of at least three replicates (* p < 0.05; ** p < 0.01).
Fig 2: lncRNA SM2 regulated the secretion of gonadotropins by TGFR1/SMAD2 signaling pathways in the pituitary cells of Hu sheep. (A) FSH and LH secretion was detected after stimulation with GnRH by ELISA. (B–D) mRNA expression of GnRHR, lncRNA SM2, SMAD2 and TGF family in pituitary cells in each group by RT-qPCR. (E) mRNA expression of TGFR1 in pituitary cells in each group by RT-qPCR. (F) Proposed model of lncRNA SM2 regulation of pituitary cell state in Hu sheep. All data are shown as the mean ± SEM of at least three replicates (* p < 0.05; ** p < 0.01).
Fig 3: LncSM2 knockdown inhibited the secretion of gonadotropins in pituitary cells. (A) Morphology of pituitary cells under a microscope. The left picture shows F1 generation pituitary cells, and the right picture shows F2 generation pituitary cells. (B) Identification of sheep pituitary cells by FSHβ and LHβ marker gene immunofluorescence staining. Scale bar, 100 μm. (C) mRNA expression of lncRNA SM2 in sheep pituitary cells treated with si-lncRNA SM2-2592, si-lncRNA SM2-3415 and si-lncRNA SM2-3794 was determined by qPCR. (D) FSH and LH secretion was detected by ELISA. (E,F) mRNA and protein expression of FSHβ and LHβ in pituitary cells in each group. All data are shown as the mean ± SEM of at least three replicates (* p < 0.05; ** p < 0.01).
Fig 4: Effect of E2 and high-dose DHT supplementation on gonadotropin subunit gene expression after OVX. (a) Cell lysates (30 μg protein) from the anterior pituitary in sham-operated (control), OVX, OVX + E2, and OVX + DHT (25 mg/kg body weight/day) rats were analyzed by SDS-PAGE followed by immunoblotting and incubation with antibody against FSH. β-actin was detected as an internal control. The bands were visualized using HRP-conjugated secondary antibody. (b) Scanning densitometry of the visualized bands using NIH ImageJ software was performed to determine differences in protein expression, normalized to that of β-actin. ∗∗P < 0.01 vs. control. FSH protein expression (E) differed significantly (P < 0.01) between OVX and OVX + E2 and OVX + DHT (25 mg/kg body weight/day) rats.
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