Novel Insight into the Potential Role of Acylglycerophosphate Acyltransferases Family Members on Triacylglycerols Synthesis in Buffalo.

Acylglycerophosphate acyltransferases (AGPATs) are the rate-limiting enzymes for the de novo pathway of triacylglycerols (TAG) synthesis. Although AGPATs have been extensively explored by evolution, expression and functional studies, little is known on functional characterization of how many members of the AGPAT family are involved in TAG synthesis and their impact on the cell proliferation and apoptosis. Here, 13 AGPAT genes in buffalo were identified, of which 12 AGPAT gene pairs were orthologous between buffalo and cattle. Comparative transcriptomic analysis and real-time quantitative reverse transcription PCR (qRT-PCR) further showed that both AGPAT1 and AGPAT6 were highly expressed in milk samples of buffalo and cattle during lactation. Knockdown of AGPAT1 or AGPAT6 significantly decreased the TAG content of buffalo mammary epithelial cells (BuMECs) and bovine mammary epithelial cells (BoMECs) by regulating lipogenic gene expression (p < 0.05). Knockdown of AGPAT1 or AGPAT6 inhibited proliferation and apoptosis of BuMECs through the expression of marker genes associated with the proliferation and apoptosis (p < 0.05). Our data confirmed that both AGPAT1 and AGPAT6 could regulate TAG synthesis and growth of mammary epithelial cells in buffalo. These findings will have important implications for understanding the role of the AGPAT gene in buffalo milk performance.

Apelin and Apelin Receptor in Follicular Granulosa Cells of Buffalo Ovaries: Expression and Regulation of Steroidogenesis.

Apelin (APLN), as a ligand for APJ (an orphan G-protein-coupled receptor), is an adipokine with pleiotropic effects in many physiological processes of the body. It has an important role in the control of reproduction particularly in females (mainly in control of ovarian function). This study was carried out to investigate the mRNA and protein amounts of APLN/APJ in granulose cells (GCs) of ovarian follicles with small (SF), medium (MF), and large (LF) sizes of buffalo (Bubalus bubalis) and the effect of IGF1 and follicle-stimulating hormone (FSH) on the expression levels of APLN/APJ. In addition, we evaluated the effect of various doses of APLN (isoforms -13 and -17) singly or in combination with IGF1 and FSH on estradiol (E2) and progesterone (P4) secretion in GCs. The mRNA and protein abundance of APLN was the highest in GCs of LF while the APJ expression enhanced with follicle enlargement in GCs (p-value <0.01). IGF1 and FSH elevated the mRNA and protein amounts of APLN and FSH, and IGF1 increased the expression of APJ in buffalo GCs (p-value <0.01). Both isoforms of APLN (-13/-17) singly or in the presence of IGF1 or FSH increased the secretion of E2 and P4 with or without preincubation of cells with APJ antagonist (ML221 10 µM), although we had some variation in the effects. Concurrently, APLN-13/-17 significantly increased the mRNA and protein expression of CYP19A1 and StAR (p-value <0.01). ML221 substantially diminished the secretion of E2 and P4 and also the expression of CY19A1 and StAR in buffalo GCs (p-value <0.01). We also revealed that APLN-13/-17 (10-9 M), singly or in response to IGF1 and FSH, increased the production of E2 and P4 in different times of stimulation. In conclusion, APLN may play a crucial role in steroidogenesis and follicular development in ovarian GCs of buffalo.

Global transcriptome analysis of different stages of preimplantation embryo development in river buffalo.

BACKGROUND: Water buffalo (Bubalus bubalis) are divided into river buffalo and swamp buffalo subspecies and are essential livestock for agriculture and the local economy. Studies on buffalo reproduction have primarily focused on optimal fertility and embryonic mortality. There is currently limited knowledge on buffalo embryonic development, especially during the preimplantation period. Assembly of the river buffalo genome offers a reference for omics studies and facilitates transcriptomic analysis of preimplantation embryo development (PED). METHODS: We revealed transcriptomic profile of four stages (2-cell, 8-cell, Morula and Blastocyst) of PED via RNA-seq (Illumina HiSeq4000). Each stage comprised three biological replicates. The data were analyzed according to the basic RNA-seq analysis process. Ingenuity analysis of cell lineage control, especially transcription factor (TF) regulatory networks, was also performed. RESULTS: A total of 21,519 expressed genes and 67,298 transcripts were predicted from approximately 81.94 Gb of raw data. Analysis of transcriptome-wide expression, gene coexpression networks, and differentially expressed genes (DEGs) allowed for the characterization of gene-specific expression levels and relationships for each stage. The expression patterns of TFs, such as POU5F1, TEAD4, CDX4 and GATAs, were elucidated across diverse time series; most TF expression levels were increased during the blastocyst stage, during which time cell differentiation is initiated. All of these TFs were involved in the composition of the regulatory networks that precisely specify cell fate. These findings offer a deeper understanding of PED at the transcriptional level in the river buffalo.