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The mammalian oviduct is crucial for the gamete maturation and supporting the early embryo development. The in vitro culturing of bovine oviductal epithelial cells (BOEC) allows researchers to study these cells without the systemic variation that occur in animals. Various in vitro culturing conditions require careful optimization.
The pathophysiological mechanisms of bacterial species in the bovine reproductive tract leading to postpartum diseases and sub-optimal fertility have become an important topic for research. Trueperella pyogenes is a commonly isolated pathogen highly associated with abnormal vaginal discharge in infected animals. Other bacterial species such as Bacillus pumilus could be involved in the development of postpartum diseases. Little is known about pathogens or their endotoxins interaction with BOEC. The present work was set up to understand cellular responses of BOEC under different culturing conditions and further in co-culture with the mentioned bacteria.
In the first part of the project, oviducts were collected at the slaughterhouse and classified into non-luteal and luteal stage. Monolayer culture of BOEC was conducted to determine cellular transcription and functional capability of BOEC 1) in transition from in vivo state to in vitro state; 2) during three consecutive cell culture passages; 3) affected by the impact of LOW (5 mM) and HIGH (25 mM) glucose content media; and 4) influenced by the effect of different phases of the estrous cycle in vitro. Total RNA was extracted from in vivo and in vitro samples and subjected to quantitative polymerase chain reaction. Moreover, collected supernatants from passage 0 (P0) and passage 3 (P3) BOEC were measured for prostaglandin E2 (PGE2), oviduct-specific glycoprotein 1 (OVGP1) and interleukin (IL) 8 release to observe the influence of in vitro culturing on BOEC functionality. Almost all candidate genes mRNA expression pattern (prostaglandin synthases, cell metabolism enzymes and mucins) were evidently changed from in vivo to in vitro. The mRNA expression pattern of microsomal prostaglandin E2 synthase 2 (PTGES2), mucin 1 (MUC1), -4 and OVGP1 was influenced by the number of cell culture passages. The mRNA expression of most candidate genes was not affected by the concentration of glucose in the culture media. The estrous cycle stage altered candidate genes mRNA expression in BOEC in vitro, notably at later passages (>P2), but not in vivo. MUC1 was the only selected gene whose mRNA expression pattern was influenced by estrous cycle stage at P1. The release rate of PGE2 and OVGP1 between P0 and P3 cells was not significantly different, yet BOEC in P3 released evident higher amounts of IL8 compared with cells in P0.
The second part of the project entirely focused on inflammatory responses of in vitro cultured BOEC under the standardized milieu, based on the outcome from the initial phase of the project, during incubation with either T. pyogenes or B. pumilus. Trypan blue staining determined BOEC viability during 24 h co-culture with different multiplicity of infection (MOI) of T. pyogenes or B. pumilus. Cells remained viable with T. pyogenes at a MOI of 0.01 and with B. pumilus at a MOI 1 and 10. Transcription level and released rate of candidate pro-inflammatory factors was not evidently changed in BOEC co-cultured with T. pyogenes compared with the controls. Meanwhile, higher mRNA expression of IL1A, -1B, -6, tumor necrosis factor alpha (TNFA), chemokine (C-X-C motif) ligand (CXCL) 1/2, -3, -5 and IL8 along with PG synthesis enzymes in BOEC co-cultured with B. pumilus was observed. Addition of B. pumilus released higher amount of IL8 and PGE2 from BOEC than controls. The impact of culturing condition on cellular immune response was followed up and it was noticed that viability and early pro-inflammatory response of P3 BOEC incubated with bacteria was clearly lower than from P0 BOEC.
In conclusion, the present work showed that conventional oviductal cell culturing highly influences the physiological and immunological responses of the cells prior to any treatment. This is very important and has to be considered during research on BOEC at in vitro level. Furthermore, this work successfully revealed a differential inflammatory response of oviductal cells against live strains of pathogenic/potential pathogenic bacteria isolated from the bovine reproductive tract. These results can contribute to a better understanding of these microorganisms interactions with the oviductal cells.