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

Sai Kumar,Bosco Jose,Meeti Punetha,Jaya Bharati,Kristin Whitworth,Mihir Sarkar,Arvind Sonwane,Shivani Khanna,Jonathan A. Green

doi:10.1038/s41598-020-77596-9

Sai Kumar, Bosco Jose + Show 7 more

Open Access

https://doi.org/10.1038/s41598-020-77596-9

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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.

Highlights

Cyclical production of fertilizable ova and steroid hormones are the two major functions performed by mammalian ovary[1]
We hypothesised that modulation of Bone morphogenetic proteins (BMPs) receptors (BMPRs)-IB gene may alter the granulosa cell function in goat similar to what was found in sheep breeds carrying the FecB mutation
The relative fold change of BMPR-IB mRNA was significantly higher in large follicles, followed by medium follicles (p < 0.05; Fig. 2B)

Summary

Introduction

Cyclical production of fertilizable ova and steroid hormones are the two major functions performed by mammalian ovary[1]. The canonical Smad dependent pathway recruit Smads as major signal transducers for the serine/threonine kinase receptors in BMP signaling. Regarded as a loss of function mutation, FecB found to exert profound effect on litter size and ovulation rate in Booroola Merino s heep[14,15]. FecB mutation induces precocious maturation of follicles, increased responsiveness to FSH and progesterone production[8]. Augmenting the reproductive efficiency of low prolific breeds is need of the hour to counter the dwindling numbers and amass goat population that leads to a subsequent increase in Chevon production. We hypothesised that modulation of BMPR-IB gene may alter the granulosa cell function in goat similar to what was found in sheep breeds carrying the FecB mutation. The current study was designed to investigate the effects of BMPR-IB gene modulation on granulosa cell function in terms of steroidogenesis, gonadotropin sensitivity and cell survivability

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Results

Conclusion