Regulation of steroidogenic function of luteal cells by thrombospondin and insulin in water buffalo (Bubalus bubalis)

Abstract

The present study examined the effect of exogenous thrombospondin 1 (TSP1) on the steroidogenic function of luteal cells cultured invitro. Furthermore, the transcriptional interaction of insulin with TSP1 and its receptor, cluster of differentiation 36 (CD36) were also investigated. At the highest dose (500ngmL−1) TSP1 significantly downregulated the expression of the angiogenic marker von Willebrand factor (vWF) and progesterone production in cultured luteal cells. Moreover, the simultaneous upregulation in the expression of caspase 3 by exogenous TSP1 was consistent with a reduction in the number of viable luteal cells as determined by 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltertrazolium bromide (MTT) assay after 72h of culture. However, the expression of critical enzymes in the progesterone synthetic pathway was not significantly modulated by treatment with TSP1 in cultured luteal cells. Knocking out of endogenous TSP1 with the clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPRassociated protein9 (Cas9) system improved the viability of luteal cells as well as increasing progesterone production and decreasing caspase 3 activation. Insulin treatment suppressed the expression of TSP1 and CD36 in cultured luteal cells in a dose- and time-dependent manner. To conclude, TSP1 acts as a negative endogenous regulator of angiogenesis that attenuates progesterone production, possibly by reducing the number of luteal cells via apoptosis during luteal regression, whereas insulin as a luteinising signal may have inhibited the thrombospondin system for the efficient development of luteal function.

Deciphering the functional role of EGR1 in Prostaglandin F2 alpha induced luteal regression applying CRISPR in corpus luteum of buffalo

“…The luteal cells were cultured by using a pre-established protocol from our laboratory [24]. In brief, mid stage CLs were excised from the ovary and were chopped using BP blades (Bard-Parker Surgical Blade).…”

“…Production of EGR1 KO luteal cells was carried out by CRISPR/Cas9 by the method described earlier [24,26]. The CRISPR/Cas9 components (single guide RNA and Cas9) were delivered via lipofection into the luteal cells GeneArt Genomic Cleavage Detection Kit (Invitrogen) was used to validate the EGR1 KO and the cleavage efficiency was calculated by the following formula [27]: Cleavage efficiency = [(sum of cleaved band intensities)/ (sum of cleaved and parental band intensities)] × 100%…”

“…The primers EGR1, AKT, Caspase3, StAR, TGFβ1, 3βHSD, CYP11A1 were designed using Gene Tool (online trial version), DNAStar (online trial version), and Oligo Analyser (open access tool) software. For the study some published were used which includes 40S ribosomal protein S15 (RPS15A) [24] and von willebrand factor (vWF). In Table 1 is given the list of primers used for the analysis.…”

Early growth response gene mediates in VEGF and FGF signaling as dissected by CRISPR in corpus luteum of water buffalo

“…Tissue collection, luteal cell culture and treatment with VEGFA and FGF2. The luteal cells were cultured using the pre-established protocol 44 . In brief, mid stage CLs were removed from the ovary with all connective tissue; and CLs were then sliced up using BP blades (Bard-Parker Surgical Blade).…”

“…primers EGR 1, EGR 2, EGR 3, EGR 4, AKT, BAX, StAR, 3βHSD and CYP11A1 were designed using DNAStar (online trial version, DNASTARLasergene 6, 2004), Gene Tool (online trial version, 2004) and Oligo Analyser 3.1 (open access tool, 2017) software. Published primers were used for 40 S ribosomal protein S15 (RPS15A)44 and vWF46 . Details of the primers used are presented inTable 1.…”