Samie Kosar Abbasi, Kowalewski Mariusz P, Schuler Gerhard, Gastal Gustavo D A, Bollwein Heinrich, Scarlet Dragos
Institute of Veterinary Anatomy, Vetsuisse Faculty Zurich, Winterthurerstrasse 260, Zurich, 8057, Switzerland.
Veterinary Clinic for Reproductive Medicine and Neonatology, Justus-Liebig-University, Frankfurter Strasse 106, 35392, Giessen, Germany.
BMC Vet Res. 2025 Apr 27;21(1):292. doi: 10.1186/s12917-025-04744-6.
In horses, the mechanisms behind ovarian follicle growth and oocyte maturation remain largely unknown. In other species, oocyte-secreted factors growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) have been related to the acquisition of developmental competence and to interaction with granulosa cells for the regulation of follicle development. This study assessed the expression and localization of GDF9 in the equine ovary, and its possible relationship with granulosa cell function.
Using custom-made antibodies, GDF9 protein was localized in oocytes from the primary follicle stage onwards. Together with BMP15, its intrafollicular concentration was higher in small antral follicles compared to larger ones (P < 0.05). Negative correlations were observed between intrafollicular BMP15 concentration and estradiol sulfate (E2S) (r = -0.36, P = 0.048), as well as between BMP15 and E2S/P4 ratio (r = -0.37, P = 0.046). In vivo, equine granulosa cells showed increasing mRNA expression of genes involved in steroidogenesis (STAR and HSD3B2) and cell proliferation (KI67) with increasing follicle size, while expression of GDF9 and of apoptosis-related genes (BCL2 and CASP3) were not affected by follicle size. Simultaneous stimulation of granulosa cells in vitro with IGF1 and cortisol significantly increased HSD3B2 and CYP19A1 transcriptional levels, as well as E2 concentration in culture media, while IGF1-induced P4 secretion was suppressed in the presence of cortisol. Blocking the stimulatory effect of IGF1 on E2, E2S and P4 by H89 was associated with increased GDF9 mRNA levels and reduced STAR, PCNA, KI67 and BCL2 mRNA expression. Significant negative correlations of GDF9 with STAR and PCNA mRNA, respectively, were seen in vivo and in vitro.
Together, our results show GDF9 localization and expression in the equine ovary and a temporal relationship with steroidogenesis and cell proliferation within the surrounding granulosa cells. Moreover, results of the in vitro study suggest a supporting role of cortisol during follicle maturation. Our study sheds light on possible mechanisms for the regulation of ovarian function in horses using GDF9.
在马中，卵巢卵泡生长和卵母细胞成熟背后的机制在很大程度上仍然未知。在其他物种中，卵母细胞分泌的因子生长分化因子9（GDF9）和骨形态发生蛋白15（BMP15）与发育能力的获得以及与颗粒细胞相互作用以调节卵泡发育有关。本研究评估了GDF9在马卵巢中的表达和定位，及其与颗粒细胞功能的可能关系。
使用定制抗体，GDF9蛋白从初级卵泡阶段开始就在卵母细胞中定位。与BMP15一起，其在小腔卵泡中的卵泡内浓度高于大卵泡（P < 0.05）。卵泡内BMP15浓度与硫酸雌二醇（E2S）之间呈负相关（r = -0.36，P = 0.048），以及BMP15与E2S/P4比值之间呈负相关（r = -0.37，P = 0.046）。在体内，随着卵泡大小增加，马颗粒细胞中参与类固醇生成（STAR和HSD3B2）和细胞增殖（KI67）的基因mRNA表达增加，而GDF9和凋亡相关基因（BCL2和CASP3）的表达不受卵泡大小影响。在体外，用IGF1和皮质醇同时刺激颗粒细胞显著增加了HSD3B2和CYP19A1转录水平以及培养基中的E2浓度，而在皮质醇存在下IGF1诱导的P4分泌受到抑制。用H89阻断IGF1对E2、E2S和P4的刺激作用与GDF9 mRNA水平增加以及STAR、PCNA、KI67和BCL2 mRNA表达降低有关。在体内和体外分别观察到GDF9与STAR和PCNA mRNA之间存在显著负相关。
总之，我们的结果显示了GDF9在马卵巢中的定位和表达，以及与周围颗粒细胞中类固醇生成和细胞增殖的时间关系。此外，体外研究结果表明皮质醇在卵泡成熟过程中起支持作用。我们的研究揭示了使用GDF9调节马卵巢功能的可能机制。
