Modulation of Granulosa Cell Function Via CRISPR-Cas Fuelled Editing of BMPR-IB Gene in Goats (Capra Hircus)

Overview

Journal Sci Rep

Specialty Science

Date 2020 Nov 25

PMID 33235250

Citations 6

Authors

Sai Kumar

Meeti Punetha

Bosco Jose

Jaya Bharati

Shivani Khanna

Arvind Sonwane

Jonathan A Green

Kristin Whitworth

Mihir Sarkar

Affiliations

Soon will be listed here.

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.

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