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

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.


Methods
All methods and experimental protocols were carried out in accordance with relevant safety guidelines and regulations.

Isolation of granulosa cells (GC).
Ovaries from apparently healthy does without any abnormalities in the reproductive tract were collected from the local slaughterhouse and transported to the laboratory at 37 °C in sterile PBS supplemented with antibiotic-antimycotic solution (Penicillin, Streptomycin and Amphotericin-B). Up on gross examination of the follicles contour and vascularity, healthy follicles were chosen and classified in to three categories based upon their diameter as Small (< 3 mm), Medium (3-5 mm) and large (> 5 mm) [22][23][24] . Granulosa cells from each follicle class (n = 6) were harvested according to the earlier described protocol 25 .
RNA extraction and cDNA synthesis. Total RNA was extracted from different GC samples using Trizin reagent (GCC Biotech) according to the manufacturer's protocol. Followed by RNA extraction, the RNA samples were subjected to RNase free DNase treatment with subsequent incubation at 56 °C for 10 min to inactivate DNAase. The quality and integrity of the RNA samples was checked by A260/A280 ratio using a Nanodrop spectrophotometer along with visualising 28S and 18S bands using agarose gel electrophoresis. 0.5 µg of total RNA was reverse transcribed to yield cDNA using RevertAid First cDNA synthesis kit (ThermoFisher Scientific).  Table 1. Annealing temperature of each primer was optimised using end point PCR. qPCR analysis. Quantitative real-time PCR was performed using the Maxima SYBR Green qPCR kit (Thermo Scientific). The slopes obtained by the amplification of a standardized dilution series were used for determining Real time PCR efficiencies. A generalised qPCR protocol was followed with initial denaturation at 95 °C for 10 min followed by 40 cycles of denaturation at 95 °C for 15 s, with primer specific annealing temperature step and extension at 72 °C for 30 s. A no template control reaction was set up for each primer to monitor the contamination and primer-dimer formation. The cycle threshold values (C t ), amplification plot and melt curves were analysed using the Biorad CFX manager Real-Time qPCR software.

Experiment 2
To demonstrate the effect of knocking out of BMPR-IB gene on granulosa cell function in goats.
Tissue collection and granulosa cell (GC) culture. A primary granulosa cell culture from large antral follicles (> 5 mm in diameter) according to the established protocol 26 . Briefly, the follicular fluid (FF) was aspirated using a sterile needle (22 gauge) and syringe. Then, the contents were transferred into a Petri plate (60 mm) containing 1X PBS followed by the removal of all cumulus-oocyte complexes (COCs). The remaining cells in the PBS were transferred into a 15 mL centrifuge tube followed by centrifugation at 300 g for 5 min. The supernatant was discarded and the GC pellet obtained was again subjected to a second wash with 1X PBS and centrifuged. www.nature.com/scientificreports/ The GC pellet was then resuspended in Dulbecco's modified Eagle's medium (DMEM) (Hyclone) containing 10% fetal bovine serum (FBS) and antibiotic-antimycotic solution. Trypan blue exclusion dye technique was used to assess the vitality of the cells. Finally, then the granulosa cells were seeded at a density of 2 × 10 5 viable cells/well in a 24-well plate and cultured in a humidified CO 2 (5%) incubator at 37 °C (Fig. 1A).

Production of BMPR-IB knock out (KO) granulosa cells (GC).
Knocking out of BMPR-IB gene in granulosa cells (Fig. 1B) was accomplished by using CRISPR-Cas genome editing technology. The CRISPR-Cas machinery was delivered into the cells using lipofection and the workflow was described below: Preparation of synthetic single guide RNA (SgRNA). SgRNA for targeting the BMPR-IB gene was designed in silico using available software. The synthesis of SgRNA was performed according to the laboratory protocol established earlier 27,28 . Prior to the SgRNA synthesis, T7 promotor sequence was added to the forward primer and the DNA template for SgRNA was generated by PCR amplification using the designed primers. Then, the SgRNA was obtained by performing in vitro transcription (IVT) using High TranscriptAid Enzyme Mix (Invitrogen) and later the in vitro transcribed SgRNA was purified using the GeneJET purification. The quality of the SgRNA was assessed by agarose gel electrophoresis and quantified using Nanodrop spectrophotometry.

Production of BMPR-IB Knock-in (KI) granulosa cells and detection. The FecB mutation in BMPR-
IB gene was introduced using Easi-CRISPR 31 . The SgRNA used earlier in the knock out experiment along with the ssODN template having the desired point mutation were used for knocking-in.
Preparation of single-stranded oligo deoxynucleotides (ssODN) containing Booroola (FecB) mutation. Single-stranded oligo deoxynucleotides (ssODN) containing Booroola (FecB) mutation (~ A746G) was designed and used as a HDR template. The ssODN with the desired mutation was flanked by two homologous arms (arm length 30-60 bases) on each side. An additional synonymous mutation (C → A), 1 bp upstream of the target site, was designed in the synthetic ssODN to identify the desired change by genomic cleavage detection assay.  www.nature.com/scientificreports/ by Tukey's honestly significant difference (HSD) test. The relative quantification of mRNA was obtained using Pfaffl's method 32 . Differences were considered significant at p < 0.05.

Results
Transcriptional abundance of BMPRs in caprine granulosa cells. Transcripts of BMPR-IA were significantly upregulated in medium and large antral follicles as compared to the small antral follicle (p < 0.05; Fig. 2A). Whereas, the relative fold change of BMPR-IB mRNA was significantly higher in large follicles, followed by medium follicles (p < 0.05; Fig. 2B). BMPR-II was significantly upregulated in the medium stage follicle and a significant downregulation in the large antral follicles was observed (p < 0.05; Fig. 2C). Overall, all the three variants of BMP receptor system were being expressed in the granulosa cells of goats.  www.nature.com/scientificreports/ significant at 72 h, has been observed in the wild cells stimulated with BMP-4(p < 0.05; Fig. 4C). The aromatase (CYP19A1) expression in WT cells was on an average 3 folds higher in and significantly differ from the other groups including the KO cells supplemented with BMP-4 (p < 0.05; Fig. 4D). Expression analysis of LHR revealed a significant increase in BMP-4 stimulated KO cells, a significant down regulation was seen in WT cells treated with BMP-4 at 72 h (p < 0.05; Fig. 4E). FSHR transcripts in BMP-4 treated WT cells were on a decreasing trend, Effects on granulosa cell steroidogenic pathway and gonadotropin sensitivity. Wild cells treated with either of the BMP ligands have shown a significant decrease in the expression StAR transcripts. On the other hand, the expression of the same is twofold higher in knock out cells stimulated with BMP-4/7 (p < 0.05; Fig. 7A,B). The P450scc (CYP11A1) too has followed the same trend as that of StAR with a highest and lowest expression being in BMP treated knock in cells and wild cells respectively (p < 0.05; Fig. 7C,D). The 3βHSD mRNA expression analysis revealed a significant up regulation in knock in cells co-cultured with a BMP ligand (BMP4/7) in comparison to untreated knock in or wild cells (p < 0.05; Fig. 7E,F). However, a significant reduction was seen in wild cells cultured with a BMP ligand. Aromatase was significantly expressed to a higher extent in BMP-4/7 treated wild cells. Wherein, its expression was least in BMP ligand stimulated granulosa cells having the FecB mutation and is around 0.5 fold less than that of untreated knock in or wild cells (p < 0.05; Fig. 8A,B). Overall, the expression analysis of the above genes in knock-in cells treated with BMP-4/7 has followed a similar pattern when compared with BMPR-IB KO cells treated with BMP-4. A significant decrease and increase in the FSHR transcripts was observed in WT cells cultured with BMP-4 and BMP-7 respectively. Whereas, the FSHR transcripts were significantly upregulated in KI cells stimulated with BMP-4/7 (p < 0.05; Fig. 8C,D). A significant downregulation of LHR gene in WT cells stimulated with either of the BMPs was observed along with a significant down regulation (p < 0.05; Fig. 8E,F).  www.nature.com/scientificreports/ Effects on cell proliferation and viability. A significant higher abundance of Caspase3 mRNA with a concurrent down regulation of PCNA (p < 0.05; Fig. 9A,B) was found in BMP treated KI cells. On the other side, Caspase 3 was down regulated (p < 0.05; Fig. 9C,D) and PCNA was up regulated in WT cells that have been supplemented with BMP-4 or BMP-7. MTT assay revealed a significant reduction of cell viability of KI cells with BMP stimulation. The cell viability of untreated knock in cells is similar to that of wild cells without BMP treatment. On the other side, there is a significant increase in the viable cell number in wild cells (p < 0.05; Fig. 9E,F) that have been supplemented with BMP-4/7 viz almost 130% in comparison to untreated wild or knock-in cells.

Effects of BMPR-IB gene KO on granulosa cells. Effects on
Effect on estradiol (E2) and progesterone (P4) production. There was a significant increase in the estradiol and decrease in the progesterone concentrations in spent media of WT cells cultured with BMP-4/7. Knock-in cells stimulated with BMP-4 or BMP-7 has significantly altered levels of E2 (p < 0.05; Fig. 10A,B) and P4 (p < 0.05; Fig. 10C,D) production in comparison to BMP treated wild cells or untreated knock in cells.

Discussion
The complex regulation of follicular dynamics dictates the degree of prolificacy in different species, thus accreting adequate knowledge in this area is necessary to augment livestock production. Follicular development involves rigid intraovarian control mechanisms in conjunction with systemic signals culminating in a series of steps mainly the recruitment, selection and growth of follicles from the primordial stage through to ovulation and corpus luteum formation 33 . Oocyte-somatic cell interactions are now widely accepted to be critical for the development of a follicle as a whole and critical for the prevention of luteinization by promoting growth and regulating steroidogenesis. With a fully established BMP system in the mammalian ovary, mutations in the BMPR-IB, BMP15 and GDF9 genes that have additive effect on ovulation rate have been discovered in various sheep breeds across the world 34 . However, none of these mutations were found in goats 35 . Hence, in our current study, we attempted to document the expression of BMP receptors and the effects of modulating BMPR-IB gene on granulosa cell function in goats. In our study, we have characterised the expression of BMP receptors (BMPRs) in granulosa cells from antral follicles of various diameters in goat. We found that all the BMP receptors viz. BMPR-IA, BMPR-IB and BMPR-II www.nature.com/scientificreports/ www.nature.com/scientificreports/ www.nature.com/scientificreports/ were expressed in granulosa cells derived from antral follicles of different sizes in regulated manner with stage specific differences. Our result was consistent with the findings of Silva et al. 36 , reported the expression of BMPRs in granulosa cells of antral follicles in goat. Significantly (p < 0.05) higher abundance of BMPR-IB mRNA in granulosa cells obtained from large antral follicles was found in our study is in accordance to earlier reports in goat, pig and buffalo [37][38][39] . The expression of BMPRs in the follicle suggests the potential role of BMP system in affecting the steroidogenesis and functional differentiation of granulosa cells. Bone morphogenetic proteins (BMPs), belonging to TGF-β superfamily play multiple roles in regulating cellular growth, differentiation and apoptosis in a wide variety of tissues. BMP-4 and BMP-7 are well-documented paracrine/autocrine modulators of granulosa cell steroidogenesis, peptide secretion and proliferation 39,40 . Hence, we have chosen BMP-4 and BMP-7 as ligands in our in vitro study to investigate the outcomes of BMPR-IB gene knock out or knock-in on granulosa cell function.
To determine the effects of BMPR-IB disruption on granulosa cell function, we have successfully knocked out BMPR-IB gene using CRIPSR-Cas genome editing technology and stimulated with BMP-4. Our results on R-Smad expression surprisingly showed a higher transcriptional abundance in knock out cells treated with the BMP ligand in comparison to BMP-4 stimulated control group over the 72 h period. In an earlier study, the deletion of BMPR-IB resulted in an increased level of p-SMAD1/5/9 up on BMP stimulation and have an equally efficient nuclear translocation in pre-osteoblasts in mice 41 . Furthermore, our study revealed a decreasing trend in the expression of StAR, CYP11A, and 3βHSD mRNA in WT cells stimulated with BMP-4, being reversed in knock out cells with BMP-4 treatment. These results are in harmony with the findings that BMP-4 inhibits progesterone secretion by inhibiting StAR and CYP11A1 (P450scc) in granulosa cells of ovine 42 . On the www.nature.com/scientificreports/ other hand, we found a significant increase in the aromatase expression in BMP-4 treated WT cells, which was abolished by knocking out of BMPR-IB gene. Yi et al. 43 , reported a reduction in levels of aromatase (CYP19A1) production in granulosa cells of mice deficient with BMPR-IB gene. In addition, increased levels progesterone (P4) and lower estradiol (E2) levels in the spent media of knock out cells stimulated with BMP-4 are in agreement with the expression pattern of StAR, CYP11A1, 3βHSD and aromatase transcripts. Altered levels of P4 and E2 suggests that BMP-4 failed to stimulate granulosa cells due to the disruption in the BMPR-IB gene. A significant increase in expression pattern of FSHR and LHR in BMP-4 stimulated KO cells was found suggesting an altered gonadotropin sensitivity. Earlier reports suggest that BMP4 via BMPR-IB downregulates the expression StAR, CYP11A1 and FSHR genes in the granulosa cells which in turn will lead to an inhibition on progesterone synthesis and the early onset of the LH surge and ovulation 44 . With regard to cell viability, our study revealed a significant reduction in the number of viable cells in BMP-4 treated KO cells, where as there is a significant increase in the viable cell count in WT cells cultured with BMP-4. The result regarding the cell viability is in congruence with the expression pattern on pro-apoptotic Caspase3 and PCNA. Earlier report in bovine antral follicles suggest an increased cell viability in granulosa cells cultured with BMPs 40 . In another study, siRNA mediated repression of the BMPR-IB gene in porcine granulosa cells had resulted in a significant inhibition on proliferation and estradiol production, whilst inducing apoptosis 38 . As a whole, knocking out of BMPR-IB gene might have relieved the inhibitory action of BMP-4 on progesterone secretion, with a concomitant decrease in the E2, cell viability and an increased gonadotropin receptivity. In order to validate the effects of FecB mutation in granulosa cells of goats, we have also introduced the Booroola (FecB) mutation using Easi-CRISPR technology. Then, the BMPR-IB gene knock-in granulosa cells were cultured separately with BMP-4 and BMP-7. In this current study, investigation on the expression pattern of R-Smads transcripts in knock-in cells up on stimulation with both the BMP ligands revealed an upregulation which is similar to the BMP-4 stimulated knock out cells. This suggests that the introduction of Booroola mutation has resulted in a loss of function of BMPR-IB. Earlier studies reported that FecB mutation attenuates the receptor activity partially or completely leading to an altered signaling pathway resulting in high ovulation rate in sheep 45,46 . Additionally, the upregulation of StAR, P450scc and 3βHSD genes along with a significant downregulation of aromatase in the BMP-4/7 treated knock in cells is analogous to the pattern that has been found in BMP-4 treated knock out cells. In our study, high P4 concentration in spent media in either of the BMP stimulated knock-in cells is in parallel with the expression of StAR,P450scc, 3βHSD genes and P4 levels in BMPR-IB KO cells. Earlier reports in granulosa cells from FecB carrier ewes revealed a higher P4 secretion than those from non-carrier animals, suggesting FecB mutation has resulted in loss of responsiveness to the inhibitory effect of BMP-4 30 . The lower E2 levels in knock-in cells treated with BMP-4/7 found in our current investigation was comparable to that of the findings in KO cells. McNatty et al. 14 , found that oestradiol from the five or more preovulatory follicles from FecB carrier ewes is compared to one or two such follicles of non-carriers. A decrease in oestradiol and inhibin production was observed in follicles from FecB carriers 13 . In addition, our investigation to test gonadotropin sensitivity revealed an upregulation in both FSHR and LHR transcripts in granulosa cells with FecB mutation. Previous studies report that FecB mutation results in the precocious maturation of ovarian follicles by increasing the sensitivity of the follicles to FSH, developing LHR and aromatase activity earlier and ovulate smaller follicular diameters [46][47][48] . Moreover, a significant reduction in viability of the BMP treated knock-in cells was revealed in MTT assay which is in accordance with down regulated PCNA and up regulated Caspase3 transcripts. This could be due to the probable loss of receptor function in knock-in cells leading to a failure in inducing proliferative and anti-apoptotic effects of BMPs resulting in ovulatory follicles with smaller diameters. Another study with luciferase reporter, granulosa cells with FecB mutation showed no response suggesting a loss of function in the receptor activity 49 .

Conclusion
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. These findings suggest the probable effects of BMPR-IB gene modulation on granulosa cell function of goats can potentially alter their reproductive performance.