Doctoral School of Exact and Natural Sciences, Jagiellonian University, Poland; Department of Endocrinology, Institute of Zoology and Biomedical Research, Faculty of Biology, Jagiellonian University, Gronostajowa 9, 30-387 Krakow, Poland.
Department of Chemical Physics, Faculty of Chemistry, Jagiellonian University, Gronostajowa 2, 30-387 Krakow, Poland.
Reprod Biol. 2023 Dec;23(4):100818. doi: 10.1016/j.repbio.2023.100818. Epub 2023 Oct 18.
The study aimed to analyze changes in biomolecular composition of granulosa and theca interna cells of porcine ovarian follicles following in vitro treatment of vitamin D and insulin alone or in combination. Medium antral follicles (n = 4/each group) were cultured alone (C; control) or in the presence of 1α,25(OH)D (VD; 100 ng/mL) and insulin (I; 10 ng/mL) separately or in combination, VD and I (VD+I). Then paraplast-embedded ovarian follicles were used for Fourier Transform Infrared (FTIR) spectroscopy and respective histological stainings. FTIR analysis revealed changes in the content of fibrous proteins (mainly collagens) within theca interna following vitamin D and insulin co-administration that was verified by Masson's trichrome staining. Treatment-dependent differences were also observed in the secondary structure of proteins, indicating enhanced conversion to α-helices in response to vitamin D and insulin action/interaction in both follicular compartments. In the granulosa and theca interna layers, tendency to lower DNA content in the VD+I group was noted and confirmed by Fulgen's staining. Finally, altered monosaccharides production in both follicular layers was found. Based on FTIR results, it is possible to attribute the observed alterations to biological processes that could be regulated by vitamin D and insulin in the porcine ovarian follicles.
本研究旨在分析维生素 D 和胰岛素单独或联合处理后猪卵巢卵泡颗粒细胞和内膜细胞中生物分子组成的变化。将中等大小的窦前卵泡（n=4/每组）分别单独培养（C；对照）或在 1α,25(OH)D（VD；100ng/mL）和胰岛素（I；10ng/mL）存在的条件下培养，或联合培养 VD 和 I（VD+I）。然后用 Paraplast 包埋的卵巢卵泡进行傅里叶变换红外（FTIR）光谱分析和相应的组织学染色。FTIR 分析显示，维生素 D 和胰岛素联合给药后，内膜中纤维蛋白（主要是胶原蛋白）的含量发生变化，马松三色染色对此进行了验证。在蛋白质的二级结构中也观察到了治疗依赖性差异，表明在两个卵泡区中，对维生素 D 和胰岛素作用/相互作用的反应增强了向 α-螺旋的转化。在颗粒层和内膜层中，VD+I 组的 DNA 含量有降低的趋势，Fulgen 染色对此进行了确认。最后，在两个卵泡层中都发现了单糖产生的改变。基于 FTIR 结果，可以将观察到的变化归因于可能由维生素 D 和胰岛素在猪卵巢卵泡中调节的生物学过程。
