Table_1_Functional and Morphological Characterization of Small and Large Steroidogenic Luteal Cells From Domestic Cats Before and During Culture.DOCX

The current study aimed to isolate, culture and characterize small (SLC) and large (LLC) steroidogenic cells from the corpora lutea (CL) of non-pregnant domestic cats. Isolation of feline SLC was based on an enzymatic digestion of luteal tissue, whereas LLC were obtained by mechanical disruption of CL. To assess function of both cell types, progesterone secretion and mRNA expression of selected genes involved in steroid and prostaglandin synthesis were measured, as well as relative transcript abundance of hormone receptors and anti-oxidative enzymes, before and during culture. The cells were cultured for 3 or 5 days without gonadotropins. Isolated feline SLC and LLC had different sizes (12 ± 3 μm vs. 34 ± 5 μm, respectively), morphologies (amount of lipid droplets) and behaved differently in culture. SLC attached and proliferated or spread quickly, but lost their steroidogenic function during culture (significant decrease in progesterone secretion and expression of steroidogenic genes). The expression of receptors for gonadotropins and prolactin also decreased. Prostaglandin synthase (PTGS2) decreased steadily over time, whereas mRNA expression of PGE2 synthase (PGES) increased. The gene expression of anti-oxidative enzyme glutathione peroxidase 4 (GPX4), also increased during culture, but not of superoxide dismutase 1 (SOD1). In comparison to SLC, LLC did not attach to culture plates, secreted more progesterone per inoculated cells and maintained steroidogenic function during culture. Expression of prostaglandin synthases (PTGS2 and PGES) was almost non-detectable. The gene expression of hormone receptors for prostaglandin F2 alpha (PTGFR), gonadotropins (LHCHR and FSHR), and prolactin (PRLR), as well as of anti-oxidative enzymes (GPX4, SOD1), increased over time. To conclude, we successfully isolated and cultured different types of feline steroidogenic luteal cells and comprehensively characterized both isolated cell types. This knowledge can be used to better understand the CL lifecycle in felines more broadly, and the established cell cultures will provide a foundation for future studies on luteolytic and luteotrophic factors in the domestic cat, and for comparison with other feline species, particularly lynx.本研究旨在从未妊娠家猫的黄体（corpora lutea, CL）中分离、培养并鉴定小型类固醇生成细胞（small steroidogenic cells, SLC）与大型类固醇生成细胞（large steroidogenic cells, LLC）。家猫SLC的分离采用酶消化黄体组织的方法，而LLC则通过机械解离黄体获得。为评估两种细胞的功能，本研究检测了培养前后细胞的孕酮分泌水平，以及类固醇与前列腺素合成相关候选基因的mRNA表达量，同时测定了激素受体与抗氧化酶的相对转录丰度。所有细胞均在不含促性腺激素的条件下培养3天或5天。 分离得到的家猫SLC与LLC在尺寸（分别为12±3 μm与34±5 μm）、形态（脂滴含量）上存在显著差异，且培养行为亦各不相同。SLC可快速贴壁、增殖或铺展，但在培养过程中会丧失类固醇生成功能——孕酮分泌量与类固醇生成相关基因的表达量均显著下降，促性腺激素与催乳素受体的表达量同样出现下调。前列腺素合酶（prostaglandin synthase, PTGS2）的表达量随培养时间持续降低，而前列腺素E2合酶（prostaglandin E2 synthase, PGES）的mRNA表达量则有所上升。抗氧化酶谷胱甘肽过氧化物酶4（glutathione peroxidase 4, GPX4）的基因表达量在培养过程中升高，但超氧化物歧化酶1（superoxide dismutase 1, SOD1）的表达量并未出现类似变化。 与SLC相比，LLC无法贴附于培养板，单位接种细胞的孕酮分泌量更高，且培养过程中可维持类固醇生成功能。LLC的前列腺素合酶（PTGS2与PGES）表达量几乎无法检测到。前列腺素F2α受体（prostaglandin F2 alpha receptor, PTGFR）、促黄体生成素/绒毛膜促性腺激素受体（luteinizing hormone/choriogonadotropin receptor, LHCHR）、卵泡刺激素受体（follicle-stimulating hormone receptor, FSHR）与催乳素受体（prolactin receptor, PRLR）的基因表达量，以及抗氧化酶GPX4、SOD1的表达量均随培养时间上升。 综上，本研究成功分离并培养了两种不同的家猫黄体类固醇生成细胞，并对两种分离细胞进行了全面的特征鉴定。本研究所得结果有助于更全面地理解猫科动物的黄体生命周期，所建立的细胞培养体系可为后续家猫黄体溶解与黄体营养因子相关研究，以及与其他猫科动物（尤其猞猁）的对比研究奠定基础。