Milk is a natural nutraceutical produced by mammals. The nanovesicles of milk play a role in horizontal gene transfer and confer health-benefits to milk consumers. These nanovesicles contain miRNA, mRNA, and proteins which mediate the intercellular communication. In this work, we isolated and characterized the buffalo milk-derived nanovesicles by dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), scanning electron microscopy (SEM), Western probing, and Fourier transform infrared (FTIR) spectroscopy. The DLS data suggested a bimodal size distribution with one mode near 50 nm and the other around 200 nm for the nanovesicles. The NTA and SEM data also supported the size of nanovesicles within a range of 50-200 nm. The FTIR measurements of nanovesicles identified some prominent absorption bands attributable to the proteins (1300-1700 cm(-1), amide A and amide B bands), lipids (2800-3100 cm(-1)), polysaccharides, and nucleic acids (900-1200 cm(-1)). The comparative expression profiles of immune miRNA signatures (miR-15b, miR-21, miR-27b, miR-125b, miR-155, and miR-500) in nanovesicles isolated from milk, serum, and urine revealed that these miRNAs are present abundantly (P< 0.05) in milk-derived nanovesicles. Milk miRNAs (miR-21 and 500) that were also found stable under different household storage conditions indicated that these could be biologically available to milk consumers. Overall, nanovesicles are a new class of bioactive compounds from buffalo milk with high proportion of stable immune miRNAs compared to urine and plasma of same animals.

Hypoxia-inducible factor 1 (HIF1) is a heterodimeric transcription factor, consisting of a constitutively expressed β-subunit (HIF1B) and a regulated α-subunit (HIF1A). In the present study, we analyzed the HIF1 driven transcriptional activity in bovine granulosa cells (GC). Treatment of GC with FSH (follicle stimulating hormone) and IGF1 (insulin-like growth factor 1) resulted in the upregulation of HIF1A mRNA expression under normoxia. Immunohistochemistry of bovine ovarian sections showed distinct staining of HIF1A in the GC layer of different staged ovarian follicles. Suppression of HIF1 using echinomycin and gene knockdown procedures revealed that HIF1 transcriptionally regulates the genes associated with steroidogenesis (STAR, HSD3B and CYP19A1) and proliferation (CCND2 and PCNA) of GC. Further, our data suggest that CYP19A1, the key gene of estradiol production, is one of the plausible downstream targets of HIF1 in bovine GC as shown by gene expression, radioimmunoassay, and chromatin precipitation analysis. Based on these results, we propose that HIF1 driven transcriptional activity plays a crucial role in GC functionality, especially steroidogenesis and proliferation in developing bovine ovarian follicles.

High-yielding dairy cows postpartum face the challenge of negative energy balance leading to elevated free fatty acids levels in the serum and follicular fluid thus affecting the ovarian function. Here, we investigated effects of physiological concentrations of palmitic acid (PA), stearic acid (SA) and oleic acid (OA) on the viability, steroid production and gene expression in a bovine granulosa cell (GC) culture model. Treatment with individual and combined fatty acids increased the CD36 gene expression, while no significant apoptotic effects were observed. Both PA and SA significantly upregulated the expression of FSHR, LHCGR, CYP19A1, HSD3B1, CCND2 and increased 17β-estradiol (E2) production, while OA downregulated the expression of these genes and reduced E2. Interestingly, STAR was equally downregulated by all fatty acids and combination treatment. E2 was significantly reduced after combination treatment. To validate the effects of OA, in vivo growing dominant follicles (10–19 mm) were injected with bovine serum albumin (BSA) with/without conjugated OA. The follicular fluid was recovered 48 h post injection. As in our in vitro model, OA significantly reduced intrafollicular E2 concentrations. In addition, expression of CD36 was significantly up- and that of CYP19A1 and STAR significantly downregulated in antral GC recovered from aspirated follicles. The ovulation rates of OA-injected follicles tended to be reduced. Our results indicate that elevated free fatty acid concentrations specifically target functional key genes in GC both in vitro and in vivo. Suggestively, this could be a possible mechanism through which elevated free fatty acids affect folliculogenesis in dairy cows postpartum.

BackgroundBovine granulosa cell culture models are important to understand molecular mechanisms of ovarian function. Folliculogenesis and luteinization are associated with increasing density of cells and local hypoxic conditions. The current study identified two reliable housekeeping genes useful for gene normalization in granulosa cells under different in vitro conditions.MethodsDuring the current experiments cells were subjected to different biological and physical stimuli, follicle stimulating hormone, different initial cell plating density and hypoxia. Transcript abundance of seven housekeeping genes was quantified by real-time RT-PCR with co-amplification of the respective external standard.ResultsThree of the genes, GAPDH, HMBS, and HPRT1 were found to be regulated by initial cell plating density, five of them, GAPDH, HMBS, HPRT1, RPLP0 and RPS18 under hypoxic conditions, but none of them after FSH stimulation. In detail, GAPDH was up regulated, but HPRT1 and HMBS were down regulated at high density and under hypoxia. Expression of RPLP0 and RPS18 was inconsistent, but was significantly down-regulated in particular at high cell density combined with hypoxia. In contrast, TBP and B2M genes were neither regulated under different plating density conditions nor by hypoxia as they showed similar expression levels under all conditions analyzed.ConclusionsThe present data indicate that TBP and B2M are appropriate housekeeping genes for normalization of transcript abundance measured by real-time RT-PCR in granulosa cells subjected to different plating densities, oxygen concentrations and FSH stimulation.

A majority of common metabolic diseases can result in excessive lipolysis, leading to elevated levels of non-esterified fatty acids (NEFAs) in the body fluids. In females, increased NEFA levels in the follicular fluid markedly alter the functions of intrafollicular cells such as granulosa cells (GCs) and oocytes. Therefore, elevated levels of NEFAs have been suggested to be a significant player of subfertility in females of both human and economically important animal species such as cattle, buffalo, sheep, pig, chicken, and dog. However, the effects imposed by saturated and unsaturated fatty acids (SFAs and UFAs) on ovarian follicles are controversial. The present review emphasizes that SFAs induce apoptosis in granulosa and cumulus cells of ovarian follicles in different species. They further could adversely affect oocyte maturation and developmental competence. Many types of UFAs affect steroidogenesis and proliferation processes and could be detrimental for follicular cells, especially when at elevated concentrations. Interestingly, monounsaturated fatty acids (MUFAs) appear to contribute to the etiology of the polycystic ovarian syndrome (PCOS) as they were found to induce the transcription and translation of the androgenic transcription factor SOX9 while downregulating its estrogenic counterpart FOXL2 in GCs. Overall, this review presents our revised understanding of the effects of different fatty acids on the female reproductive success, which may allow other researchers and clinicians to investigate the mechanisms for treating metabolic stress-induced female infertility.