Physiology and Climatology Division, ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh, 243122, India.
Division of Animal Genetics, ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh, 243122, India.
Sci Rep. 2020 Nov 24;10(1):20446. doi: 10.1038/s41598-020-77596-9.
BMPs are multifunctional growth factors implicated in regulating the ovarian function as key intra-ovarian factors. Biological effects of BMPs are mediated through binding with membrane bound receptors like BMPR-IB and initiating downstream Smad signaling pathway. FecB mutation, regarded as a loss of function mutation in the BMPR-IB gene was identified in certain sheep breeds having high fecundity. Similar type of fecundity genes in goats have not been discovered so far. Hence, the current study was designed to investigate the effects of BMPR-IB gene modulation on granulosa cell function in goats. The BMPR-IB gene was knocked out using CRISPR-Cas technology in granulosa cells and cultured in vitro with BMP-4 stimulation for three different durations In addition, the FecB mutation was introduced in the BMPR-IB gene applying Easi-CRISPR followed by BMP-4/7 stimulation for 72 h. Steroidogenesis and cell viability were studied to explore the granulosa cell function on BMPR-IB gene modulation. BMPRs were found to be expressed stage specifically in granulosa cells of goats. Higher transcriptional abundance of R-Smads, LHR and FSHR indicating sensitisation of Smad signaling and increased gonadotropin sensitivity along with a significant reduction in the cell proliferation and viability was observed in granulosa cells upon BMPR-IB modulation. The inhibitory action of BMP-4/7 on P4 secretion was abolished in both KO and KI cells. Altogether, the study has revealed an altered Smad signaling, steroidogenesis and cell viability upon modulation of BMPR-IB gene in granulosa cells similar to that are documented in sheep breeds carrying the FecB mutation.
BMPs 是多功能生长因子，作为关键的卵巢内因子，参与调节卵巢功能。BMPs 的生物学效应通过与膜结合受体（如 BMPR-IB）结合并启动下游 Smad 信号通路来介导。在具有高繁殖力的某些绵羊品种中，发现了 BMPR-IB 基因的 FecB 突变，被认为是该基因的功能丧失突变。到目前为止，还没有发现山羊中类似的繁殖力基因。因此，本研究旨在研究 BMPR-IB 基因调节对山羊颗粒细胞功能的影响。使用 CRISPR-Cas 技术在颗粒细胞中敲除 BMPR-IB 基因，并在体外用 BMP-4 刺激培养 3 个不同的时间段。此外，通过 Easi-CRISPR 将 FecB 突变引入 BMPR-IB 基因，然后用 BMP-4/7 刺激 72 小时。研究了类固醇生成和细胞活力，以探索 BMPR-IB 基因调节对颗粒细胞功能的影响。BMPRs 在山羊颗粒细胞中表现出特定阶段的表达。在 BMPR-IB 调节后，R-Smads、LHR 和 FSHR 的转录丰度更高，表明 Smad 信号的敏感性增加，以及促性腺激素敏感性增加，同时细胞增殖和活力显著降低。在 KO 和 KI 细胞中，BMP-4/7 对 P4 分泌的抑制作用被消除。总的来说，该研究揭示了在颗粒细胞中调节 BMPR-IB 基因后，Smad 信号、类固醇生成和细胞活力发生改变，类似于携带 FecB 突变的绵羊品种中所记录的情况。
