Modulation of granulosa cell function via CRISPR-Cas fuelled editing of BMPR-IB gene in goats (Capra hircus)

Abstract

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.

Figure 1

(A) 75–80% in vitro cultured caprine granulosa cells. (B) In vitro cultured BMPR-IB KO cells. (C) Gel image of Genomic Cleavage Detection Assay of granulosa cells transfected with components of CRISPR-Cas system for BMPR-IB gene knock out (KO) using Lipofectamine 2000. Lane 1 and 2: Negative control samples for BMPR-IB KO, Lane3: Genomic cleavage showing both Parent band and Cleaved band. (D) Gel image of Genomic Cleavage Detection Assay of granulosa cells electroporated with components of CRISPR-Cas system along with ssODN for BMPR-IB gene knock-in (KI) using Lipofectamine 2000. Lane1: Genomic cleavage showing both Parent band and Cleaved band and Lane2: Negative control sample for BMPR-IB knock-in (KI).

Figure 2

Transcriptional abundance of BMPRs (BMPR-IA, BMPR-IB and BMPR-II) in granulosa cells from different antral follicles of Goat (n = 6/group). All values are shown as mean ± SEM. Different superscripts denote statistically different values (p < 0.05). Abbreviations: FL1, FL2 and FL3 denotes antral follicles of < 3 mm, 3–5 mm and > 5 mm diameters respectively.

Figure 3

Effect of BMPR-IB gene KO on expression of Smad1, Smad5 and Smad 8 transcripts in in vitro cultured granulosa cells stimulated with BMP-4 (50 ng/ml) for three different time durations (24, 48 and 72 h). All values are shown as mean ± SEM. Different superscripts denote statistically different values (p < 0.05). Abbreviations: WT indicate wild type cells, WT + BMP-4 indicate wild cells treated with BMP-4 (50 ng/mL), KO indicate BMPR-IB gene knock out cells. KO + BMP-4 indicate KO cells treated with BMP-4(50 ng/ml).

Figure 4

Effect of BMPR-IB gene KO on expression pattern of StAR, CYP11A1, 3βHSD, Aromatase, LHR and FSHR genes in in vitro cultured granulosa cells stimulated with BMP-4 (50 ng/ml) for three different time durations (24, 48 and 72 h). All values are shown as mean ± SEM. Different superscripts denote statistically different values (p < 0.05). Abbreviations: WT indicate wild type cells, WT + BMP-4 indicate wild cells treated with BMP-4 (50 ng/mL), KO indicate BMPR-IB gene knock out cells. KO + BMP-4 indicate KO cells treated with BMP-4(50 ng/ml).

Figure 5

(A, B) Effect of BMPR-IB gene KO on expression pattern of PCNA, Caspase3 genes on in vitro cultured granulosa cells stimulated with BMP-4 (50 ng/ml) for three different time durations (24, 48 and 72 h). All values are shown as mean ± SEM. Different superscripts denote statistically different values (p < 0.05). Abbreviations: WT indicate wild type cells, WT + BMP-4 indicate wild cells treated with BMP-4 (50 ng/mL), KO indicate BMPR-IB gene knock out cells. KO + BMP-4 indicate KO cells treated with BMP-4(50 ng/ml). (C, D, E) Effect of BMPR-IB gene KO on cell viability in in vitro cultured granulosa cells stimulated with BMP-4 (50 ng/ml) for three different time durations (24, 48 and 72 h) assessed by MTT assay. All values are shown as mean ± SEM. Different superscripts denote statistically different values (p < 0.05). Abbreviations: WT indicate wild type cells, WT + BMP-4 indicate wild cells treated with BMP-4 (50 ng/mL), KO indicate BMPR-IB gene knock out cells. KO + BMP-4 indicate KO cells treated with BMP-4(50 ng/ml). (F, G) Effect of BMPR-IB gene KO on Progesterone (P4) and Estradiol (E2) stimulation of BMPR-IB gene KO on All values are shown as mean ± SEM. Different superscripts denote statistically different values (p < 0.05). Abbreviations: WT indicate wild type cells, WT + BMP-4 indicate wild cells treated with BMP-4 (50 ng/mL), KO indicate BMPR-IB gene knock out cells. KO + BMP-4 indicate KO cells treated with BMP-4(50 ng/ml).

Figure 6

Effect of BMPR-IB gene KI on expression of Smad1, Smad5 and Smad 8 transcripts in in vitro cultured granulosa cells stimulated with BMP-4 (50 ng/ml) or BMP-7 (100 ng/ml) for 72 h. All values are shown as mean ± SEM. Different superscripts denote statistically different values (p < 0.05). Abbreviations: WT indicate wild type cells, WT + BMP-4/BMP-7 indicate wild cells treated with BMP-4 (50 ng/mL), KO indicate BMPR-IB gene knock out cells. KI + BMP-4/BMP-7 indicate KI cells treated with BMP-4(50 ng/ml) or BMP-7 (100 ng/ml).

Figure 7

Effect of BMPR-IB gene KI on expression of StAR, CYP11A1 and 3βHSD genes in in vitro cultured granulosa cells stimulated with BMP-4 (50 ng/ml) or BMP-7 (100 ng/ml) for 72 h. All values are shown as mean ± SEM. Different superscripts denote statistically different values (p < 0.05). Abbreviations: WT indicate wild type cells, WT + BMP-4/BMP-7 indicate wild cells treated with BMP-4 (50 ng/mL), KI indicate BMPR-IB gene knock out cells. KI + BMP-4/BMP-7 indicate KI cells treated with BMP-4(50 ng/ml) or BMP-7 (100 ng/ml).

Figure 8

Effect of BMPR-IB gene KI on expression of Aromatase, FSHR and LHR genes in in vitro cultured granulosa cells stimulated with BMP-4 (50 ng/ml) or BMP-7 (100 ng/ml) for 72 h. All values are shown as mean ± SEM. Different superscripts denote statistically different values (p < 0.05). Abbreviations: WT indicate wild type cells, WT + BMP-4/BMP-7 indicate wild cells treated with BMP-4 (50 ng/mL), KI indicate BMPR-IB gene knock out cells. KI + BMP-4/BMP-7 indicate KI cells treated with BMP-4(50 ng/ml) or BMP-7 (100 ng/ml).

Figure 9

Effect of BMPR-IB gene KI on expression pattern of PCNA, Capase3 genes and cell viability of in vitro cultured granulosa cells stimulated with BMP-4 (50 ng/ml) or BMP-7 (100 ng/ml) for 72 h. All values are shown as mean ± SEM. Different superscripts denote statistically different values (p < 0.05). Abbreviations: WT indicate wild type cells, WT + BMP-4/BMP-7 indicate wild cells treated with BMP-4 (50 ng/mL), KI indicate BMPR-IB gene knock out cells. KI + BMP-4/BMP-7 indicate KI cells treated with BMP-4(50 ng/ml) or BMP-7 (100 ng/ml).

Figure 10

Effect of BMPR-IB gene KI on P4 and E2 synthesis in in vitro cultured granulosa cells stimulated with BMP-4 (50 ng/ml) or BMP-7 (100 ng/ml) for 72 h. All values are shown as mean ± SEM. Different superscripts denote statistically different values (p < 0.05). Abbreviations: WT indicate wild type cells, WT + BMP-4/BMP-7 indicate wild cells treated with BMP-4 (50 ng/mL), KI indicate BMPR-IB gene knock out cells. KI + BMP-4/BMP-7 indicate KI cells treated with BMP-4(50 ng/ml) or BMP-7 (100 ng/ml).