Expression and functional role of IGFs during early pregnancy in placenta of water buffalo.

Adequate vascularisation is a key factor for successful fetal development. We hypothesized that Insulin-Like Growth Factor (IGF) family members regulate angiogenesis along with promoting fetal development and growth. In this experiment, we determined the expression and functional role of IGF family in placental compartments (caruncle; CAR, cotyledon; COT) during different stages of early pregnancy in the water buffalo (Bubalus bubalis). Samples were collected from early pregnancy 1 (EP1, 28-45 days), early pregnancy 2 (EP2, 45-90 days), and third stage of estrous cycle (11-16 days), which was taken as control. In addition, the role of IGF1 on mRNA expression of vWF, StAR, CYP11A1, 3ßHSD, PCNA, and BAX were elucidated in cultured trophoblast cells (TCC) obtained from EP2. Quantitative real-time PCR (q-PCR), westernblot, and immunohistochemistry were done to investigate the gene expression, protein expression, and localization of examined factors, and RIA was also done to assess progesterone (P4) concentration. Expression of IGFs, its receptors and binding proteins were found to be significantly higher (p < 0.05) in both CAR and COT as compared to control during early pregnancy, except binding proteins IGFBP1, 3 and 4 which were significantly (p < 0.05) downregulated in COT with advancement of pregnancy. mRNA expression was consistent with the findings of immunoblotting and immunolocalization experiments. Trophoblasts cell culture (TCC) study showed a significant time and dose-dependent effect of IGF1 onsteroidogenic transcript, which was found to be maximum at 100 ng/ml that paralleled with P4 accretion in the media (p < 0.05). Further, IGF1 upregulated the transcripts of vWF, PCNA, and downregulated BAX at the same concentration (p < 0.05). Overall, our results demonstrated that the expression of IGFs is a site-specific phenomenon in placentome, which indicates autocrine/paracrine and endocrine function. Our in-vitro finding support that IGF1 plays a critical role in placental development by promoting angiogenesis, steroid synthesis, and cell proliferation during early pregnancy.

Expression and functional role of fibroblast growth factors (FGF) in placenta during different stages of pregnancy in water buffalo (Bubalus bubalis).

The present study documented the expression and functional role of Fibroblast growth factors (FGFs) family and their receptors (Fibroblast growth factor receptor, FGFRs) in placenta (Cotyledon; COT, Caruncle; CAR) during different stages of pregnancy in water buffalo. Samples were collected from Early pregnancy 1 (EP1); Early pregnancy 2 (EP2); Mid pregnancy (MP) and Late pregnancy (LP) while diestrus stage of oestrus cycle (NP) was taken as control. In addition, modulatory role of FGF2 on mRNA expression of von Willebrand factor (vWF), Proliferating cell nuclear antigen (PCNA), steroidogenic acute regulatory protein (StAR), cytochrome P450 cholesterol side-chain cleavage (CYP11A1), 3ß-hydroxysteroid dehydrogenase (3ßHSD) and BCL2 Associated X (BAX) were studied in cultured trophoblast cells (TCC), obtained from EP2. Real-time PCR (qPCR), Western blot, and immunohistochemistry were applied to investigate mRNA and protein expressions, and the localization of examined factors whereas, P4 secretion was assessed by RIA. The mRNA and protein expression of FGFs and its receptors were maximum (P < 0.05) during EP (EP1 and EP2) in COT. However, FGFR1 and FGFR4 were upregulated (P < 0.05) during EP2 and MP in COT. Similarly, the mRNA and protein expression of FGFs and its receptors were upregulated (P < 0.05) during all stages of pregnancy in CAR. FGF family members were localized in the cytoplasm of trophoblast cells as well as in fetal blood vessels. At 100 ng/ml dosage, FGF2 stimulated the transcript of vWF maximally (P < 0.05). P4 secretion in trophoblast cells treated with FGF2 was maximum with the highest dose at 72 h. These findings corroborate that FGF acts locally in the trophoblast cells to modulate steroid hormone viz. progesterone synthesis, promote angiogenesis and favors cell survivability indicating that this factor may play an essential role in the regulation of placental formation and function in buffalo.